技术领域technical field
本申请要求2009年5月28日提交的美国临时专利申请号61/181,773、2009年8月5日提交的美国临时专利申请号61/231,458和2009年8月28日提交的美国临时专利申请号61/237,886的优先权，它们通过引用整体并入本文。This application claims U.S. Provisional Patent Application No. 61/181,773 filed May 28, 2009, U.S. Provisional Patent Application No. 61/231,458 filed Aug. 5, 2009, and U.S. Provisional Patent Application No. 61/231,458 filed Aug. 28, 2009 61/237,886, which are hereby incorporated by reference in their entirety.
本发明的实施方案包括调节抗病毒基因和相关分子的表达和/或功能的寡核苷酸。Embodiments of the invention include oligonucleotides that modulate the expression and/or function of antiviral genes and related molecules.
背景技术Background technique
DNA-RNA和RNA-RNA杂交对于核酸功能的许多方面(包括DNA复制、转录和翻译)而言为重要的。杂交对于探测特定核酸或者改变其表达的各种技术而言亦为主要的。反义核苷酸例如通过与靶RNA杂交来破坏基因表达，从而干扰RNA剪接、转录、翻译和复制。反义DNA具有附加的特征，该特征为DNA-RNA杂合体充当核糖核酸酶H消化的底物，该活性存在于大多数细胞类型中。可将反义分子递送到细胞中，这与寡脱氧核苷酸(ODN)的情况一样，或者它们如RNA分子一样可由内源基因表达。FDA最近批准了一种反义药物，VITRAVENETM(用于治疗巨细胞病毒视网膜炎)，这反映了反义物具有治疗应用。DNA-RNA and RNA-RNA hybridization is important for many aspects of nucleic acid function, including DNA replication, transcription and translation. Hybridization is also central to various techniques that detect specific nucleic acids or alter their expression. Antisense nucleotides disrupt gene expression, for example, by hybridizing to target RNA, thereby interfering with RNA splicing, transcription, translation and replication. Antisense DNA has the added feature that DNA-RNA hybrids serve as substrates for RNase H digestion, an activity present in most cell types. Antisense molecules can be delivered into cells, as is the case with oligodeoxynucleotides (ODNs), or they can be expressed by endogenous genes, like RNA molecules. The recent FDA approval of an antisense drug, VITRAVENE(TM) (for the treatment of cytomegalovirus retinitis), reflects the therapeutic utility of antisense.
发明内容Contents of the invention
在一个实施方案中，本发明提供了通过使用靶向天然反义转录物的任何区域的反义寡核苷酸来抑制天然反义转录物的作用，引起相应有义基因的增量调节的方法。本文也考虑天然反义转录物的抑制可通过siRNA、核酶和小分子来达到，认为所述siRNA、核酶和小分子在本发明的范围之内。In one embodiment, the present invention provides a method of inhibiting the action of a natural antisense transcript, resulting in upregulation of the corresponding sense gene, by using an antisense oligonucleotide targeting any region of the natural antisense transcript . It is also contemplated herein that inhibition of natural antisense transcripts can be achieved by siRNAs, ribozymes and small molecules, which are considered to be within the scope of the invention.
一个实施方案提供了在体内或在体外调节患者细胞或组织中的抗病毒基因多核苷酸的功能和/或表达的方法，所述方法包括：使所述细胞或组织接触长度为5-30个核苷酸的反义寡核苷酸，其中所述寡核苷酸与以下多核苷酸的反向互补序列具有至少50％序列同一性：所述多核苷酸包含在SEQ ID SEQ ID NO：4的核苷酸1-1267、SEQ ID NO：5的核苷酸1-586、SEQ ID SEQ ID NO：6的核苷酸1-741、SEQ ID NO：7的核苷酸1-251、SEQ ID NO：8的核苷酸1-681和SEQ ID NO：9的核苷酸1-580之内的5-30个连续核苷酸，从而在体内或在体外调节患者细胞或组织中的抗病毒基因多核苷酸的功能和/或表达。One embodiment provides a method for regulating the function and/or expression of an antiviral gene polynucleotide in a patient's cell or tissue in vivo or in vitro, the method comprising: contacting the cell or tissue with a length of 5-30 An antisense oligonucleotide of nucleotides, wherein said oligonucleotide has at least 50% sequence identity to the reverse complement of the polynucleotide contained in SEQ ID SEQ ID NO: 4 Nucleotides 1-1267 of SEQ ID NO: 5, nucleotides 1-586 of SEQ ID NO: 5, nucleotides 1-741 of SEQ ID NO: 6, nucleotides 1-251 of SEQ ID NO: 7, SEQ ID NO: 5-30 consecutive nucleotides within nucleotides 1-681 of ID NO: 8 and nucleotides 1-580 of SEQ ID NO: 9, thereby regulating the anti-inflammatory activity in patient cells or tissues in vivo or in vitro Function and/or expression of viral gene polynucleotides.
在另一个实施方案中，寡核苷酸靶向抗病毒基因多核苷酸的天然反义序列，例如SEQ ID NO：4-9所述的核苷酸，及其任何变体、等位基因、同源物、突变体、衍生物、片段和互补序列。反义寡核苷酸的实例如SEQ ID NO：10-30所述。In another embodiment, the oligonucleotide targets the natural antisense sequence of the antiviral gene polynucleotide, such as the nucleotides set forth in SEQ ID NO: 4-9, and any variants, alleles, Homologues, mutants, derivatives, fragments and complements. Examples of antisense oligonucleotides are set forth in SEQ ID NO: 10-30.
另一个实施方案提供了在体内或在体外调节患者细胞或组织中抗病毒基因多核苷酸的功能和/或表达的方法，所述方法包括：使所述细胞或组织接触长度为5-30个核苷酸的反义寡核苷酸，其中所述寡核苷酸与抗病毒基因多核苷酸的反义物的反向互补序列具有至少50％序列同一性；从而在体内或在体外调节患者细胞或组织中抗病毒基因多核苷酸的功能和/或表达。Another embodiment provides a method for regulating the function and/or expression of an antiviral gene polynucleotide in a patient's cell or tissue in vivo or in vitro, the method comprising: contacting the cell or tissue with a length of 5-30 Antisense oligonucleotides of nucleotides, wherein said oligonucleotides have at least 50% sequence identity to the reverse complement of the antisense of the antiviral gene polynucleotides; thereby modulating the patient in vivo or in vitro Function and/or expression of antiviral gene polynucleotides in cells or tissues.
另一个实施方案提供了在体内或在体外调节患者细胞或组织中抗病毒基因多核苷酸的功能和/或表达的方法，所述方法包括：使所述细胞或组织接触长度为5-30个核苷酸的反义寡核苷酸，其中所述寡核苷酸与抗病毒基因反义多核苷酸的反义寡核苷酸具有至少50％序列同一性；从而在体内或在体外调节患者细胞或组织中抗病毒基因多核苷酸的功能和/或表达。Another embodiment provides a method for regulating the function and/or expression of an antiviral gene polynucleotide in a patient's cell or tissue in vivo or in vitro, the method comprising: contacting the cell or tissue with a length of 5-30 Antisense oligonucleotides of nucleotides, wherein said oligonucleotides have at least 50% sequence identity with antisense oligonucleotides of antiviral gene antisense polynucleotides; Function and/or expression of antiviral gene polynucleotides in cells or tissues.
在一个实施方案中，组合物包含一种或更多种与有义和/或反义抗病毒基因多核苷酸结合的反义寡核苷酸。In one embodiment, the composition comprises one or more antisense oligonucleotides conjugated to sense and/or antisense antiviral gene polynucleotides.
在另一个实施方案中，所述寡核苷酸包含一个或更多个经修饰或取代的核苷酸。In another embodiment, the oligonucleotide comprises one or more modified or substituted nucleotides.
在另一个实施方案中，所述寡核苷酸包含一个或更多个经修饰的键。In another embodiment, the oligonucleotide comprises one or more modified linkages.
在另一个实施方案中，所述经修饰的核苷酸包含经修饰的碱基，该碱基包含硫代磷酸酯、甲基膦酸酯、肽核酸、2’-O-甲基、氟或碳、亚甲基或其它锁定核酸(LNA)分子。优选地，所述经修饰的核苷酸为锁定核酸分子，包括α-L-LNA。In another embodiment, the modified nucleotides comprise modified bases comprising phosphorothioate, methylphosphonate, peptide nucleic acid, 2'-O-methyl, fluoro, or Carbon, methylene, or other locked nucleic acid (LNA) molecules. Preferably, the modified nucleotides are locked nucleic acid molecules, including α-L-LNA.
在另一个实施方案中，将所述寡核苷酸皮下地、肌肉内地、静脉内地或腹膜内地施用给患者。In another embodiment, the oligonucleotide is administered to the patient subcutaneously, intramuscularly, intravenously or intraperitoneally.
在另一个实施方案中，在药物组合物中施用所述寡核苷酸。治疗方案包括向患者施用反义化合物至少一次；然而，可将此治疗修改成包含在一段时间内的多次给药。所述治疗可与一种或更多种其它类型的疗法组合。In another embodiment, the oligonucleotide is administered in a pharmaceutical composition. The treatment regimen includes at least one administration of the antisense compound to the patient; however, this treatment can be modified to include multiple administrations over a period of time. The treatment may be combined with one or more other types of therapy.
在另一个实施方案中，将所述寡核苷酸封装到脂质体中或附着于载体分子(例如胆固醇、TAT肽)。In another embodiment, the oligonucleotides are encapsulated into liposomes or attached to carrier molecules (eg cholesterol, TAT peptide).
其它方面描述于下文。Other aspects are described below.
附图说明Description of drawings
图1是实时PCR结果的图，它显示了与对照相比，用硫代磷酸寡核苷酸处理HepG2细胞后RIG1 mRNA的倍数变化+标准差，所述硫代磷酸寡核苷酸使用脂质体转染胺2000(Lipofectamine 2000)引入。实时PCR结果表明，在用针对RIG1反义物hs.601664和AK300104设计的siRNA处理后48h，HepG2细胞中RIG1 mRNA水平显著增加。表示为CUR-0544、CUR-0546、CUR-0548、CUR-0550、CUR-0552、CUR-0554、CUR-0556、CUR-0558和CUR-0560的条分别对应用SEQ ID NO：10-18处理的样品。Figure 1 is a graph of real-time PCR results showing the fold change + standard deviation of RIG1 mRNA after treatment of HepG2 cells with phosphorothioate oligonucleotides using lipid Lipofectamine 2000 (Lipofectamine 2000) was introduced. Real-time PCR results showed that the level of RIG1 mRNA in HepG2 cells was significantly increased 48 h after treatment with siRNA designed against RIG1 antisense hs.601664 and AK300104. Bars denoted CUR-0544, CUR-0546, CUR-0548, CUR-0550, CUR-0552, CUR-0554, CUR-0556, CUR-0558 and CUR-0560 were treated with SEQ ID NO: 10-18, respectively sample.
图2是实时PCR结果的图，它显示了与对照相比，用硫代磷酸寡核苷酸处理HepG2细胞后MDA5 mRNA的倍数变化+标准差，所述硫代磷酸寡核苷酸使用脂质体转染胺2000引入。实时PCR结果表明，在用针对MDA5反义物BU663736设计的2种寡物处理后48h，HepG2细胞中MDA5 mRNA水平显著增加。表示为CUR-0908、CUR-0909、CUR-0910、CUR-0911、CUR-0912、CUR-0913、CUR-0914和CUR-0915的条分别对应用SEQ ID NO：19-26处理的样品。Figure 2 is a graph of real-time PCR results showing the fold change + standard deviation of MDA5 mRNA after treatment of HepG2 cells with phosphorothioate oligonucleotides using lipid Transfectamine 2000 was introduced. The results of real-time PCR showed that the level of MDA5 mRNA in HepG2 cells was significantly increased 48 hours after treatment with the two oligos designed against MDA5 antisense BU663736. Bars denoted CUR-0908, CUR-0909, CUR-0910, CUR-0911, CUR-0912, CUR-0913, CUR-0914 and CUR-0915 are for samples treated with SEQ ID NO: 19-26, respectively.
图3是实时PCR结果的图，它显示了与对照相比，用硫代磷酸寡核苷酸处理HepG2细胞后IFNA1 mRNA的倍数变化+标准差，所述硫代磷酸寡核苷酸使用脂质体转染胺2000引入。实时PCR结果表明，在用针对IFNAI反义物DA393812设计的1种寡物处理后48h，HepG2细胞中IFNAI mRNA水平显著增加。表示为CUR-0916、CUR-0917、CUR-0918和CUR-0919的条分别对应用SEQ ID NO：27-30处理的样品。Figure 3 is a graph of real-time PCR results showing the fold change + standard deviation of IFNA1 mRNA after treatment of HepG2 cells with phosphorothioate oligonucleotides using lipid Transfectamine 2000 was introduced. The results of real-time PCR showed that the level of IFNAI mRNA in HepG2 cells was significantly increased 48 hours after treatment with an oligo designed against IFNAI antisense DA393812. Bars denoted CUR-0916, CUR-0917, CUR-0918 and CUR-0919 correspond to samples treated with SEQ ID NO: 27-30, respectively.
图4是实时PCR结果的图，它显示了与对照相比，用硫代磷酸寡核苷酸处理ZR75细胞后IFNA1 mRNA的倍数变化+标准差，所述硫代磷酸寡核苷酸使用脂质体转染胺2000引入。实时PCR结果表明，在用针对IFNAI反义物DA393812设计的1种寡物处理后48h，ZR75细胞中IFNAI mRNA水平显著增加。表示为CUR-0916、CUR-0919、CUR-0918和CUR-0917的条分别对应用SEQ ID NO：27、30、29和28处理的样品。Figure 4 is a graph of real-time PCR results showing the fold change + standard deviation of IFNA1 mRNA after treatment of ZR75 cells with phosphorothioate oligonucleotides using lipid Transfectamine 2000 was introduced. The results of real-time PCR showed that the level of IFNAI mRNA in ZR75 cells was significantly increased 48 hours after treatment with an oligo designed against IFNAI antisense DA393812. Bars denoted CUR-0916, CUR-0919, CUR-0918 and CUR-0917 correspond to samples treated with SEQ ID NO: 27, 30, 29 and 28, respectively.
图5是实时PCR结果的图，它显示了与对照相比，用硫代磷酸寡核苷酸处理HUVEC细胞后IFNA1 mRNA的倍数变化+标准差，所述硫代磷酸寡核苷酸使用脂质体转染胺2000引入。实时PCR结果表明，在用针对IFNAI反义物DA393812设计的1种寡物处理后48h，HUVEC细胞中IFNAI mRNA水平显著增加。表示为CUR-0916、CUR-0917、CUR-0918和CUR-0919的条分别对应用SEQ ID NO：27-30处理的样品。Figure 5 is a graph of real-time PCR results showing the fold change + standard deviation of IFNA1 mRNA after treatment of HUVEC cells with phosphorothioate oligonucleotides using lipid Transfectamine 2000 was introduced. The results of real-time PCR showed that the level of IFNAI mRNA in HUVEC cells was significantly increased 48 hours after treatment with an oligo designed against IFNAI antisense DA393812. Bars denoted CUR-0916, CUR-0917, CUR-0918 and CUR-0919 correspond to samples treated with SEQ ID NO: 27-30, respectively.
图6：ELISA测定结果表明，IFNA蛋白在来自MCF7细胞的提取物中被增量调节，所述MCF7细胞用针对IFNAI反义物DA393812设计的寡物处理过。表示为CUR-0916、CUR-0919、CUR-0918和CUR-0917的条分别对应用SEQ ID NO：27、30、29和28处理的样品。Figure 6: ELISA assay results demonstrating that IFNA protein is upregulated in extracts from MCF7 cells treated with oligos designed against the IFNAI antisense DA393812. Bars denoted CUR-0916, CUR-0919, CUR-0918 and CUR-0917 correspond to samples treated with SEQ ID NO: 27, 30, 29 and 28, respectively.
序列表描述Sequence Listing Description
SEQ ID NO：1：智人无调(atonal)同系物1(果蝇属)(ATOH1)，mRNA(NCBI登记号：NM_005172)。SEQ ID NO: 1: Homo sapiens atonal homolog 1 (Drosophila) (ATOH1), mRNA (NCBI accession number: NM_005172).
SEQ ID NO：2：用解螺旋酶C结构域1(IFIH1)诱导的智人干扰素，mRNA(NCBI登记号：NM_022168.2)。SEQ ID NO: 2: Homo sapiens interferon, mRNA induced by helicase C domain 1 (IFIH1) (NCBI accession number: NM_022168.2).
SEQ ID NO：3：NM_024013.1|智人干扰素，α1(IFNA1)，mRNA(NCBI登记号：NM_005172)。SEQ ID NO: 3: NM_024013.1 | Homo sapiens interferon, alpha 1 (IFNA1), mRNA (NCBI accession number: NM_005172).
SEQ ID NO：4-9：SEQ ID NO：4：天然的RIG1反义序列(AK300104)；SEQ ID NO：5：天然的RIG1反义序列(hs.601664)；SEQ ID NO：6：天然的RIG1反义序列(hs.104091)；SEQ IDNO：7：天然的MDA5反义序列(Hs.692345)；SEQ ID NO：8：天然的MDA5反义序列(BU663736)和SEQ ID NO：9：天然的IFNA1反义序列(DA393812)SEQ ID NO: 4-9: SEQ ID NO: 4: native RIG1 antisense sequence (AK300104); SEQ ID NO: 5: native RIG1 antisense sequence (hs.601664); SEQ ID NO: 6: native RIG1 antisense sequence (hs.104091); SEQ ID NO: 7: native MDA5 antisense sequence (Hs.692345); SEQ ID NO: 8: native MDA5 antisense sequence (BU663736) and SEQ ID NO: 9: native IFNA1 antisense sequence (DA393812)
SEQ ID NO：10-30：反义寡核苷酸。‘r’表示RNA，*表示硫代磷酸酯键。SEQ ID NO: 10-30: Antisense oligonucleotides. 'r' indicates RNA, * indicates a phosphorothioate bond.
SEQ ID NO：31和39分别是反义寡核苷酸SEQ ID NO：10和18的反向互补序列。‘r’表示RNA。SEQ ID NO: 31 and 39 are the reverse complements of the antisense oligonucleotides SEQ ID NO: 10 and 18, respectively. 'r' indicates RNA.
具体实施方式detailed description
参考用于说明的示例应用在下文中描述本发明的数个方面。应当理解的是，陈述许多具体细节、关系和方法来提供对本发明的充分理解。然而，在相关领域的普通技术人员将容易地认识到，可在不含一个或更多个具体细节的情况下实施本发明或者可用其它方法来实施本发明。本发明不受行为或事件的排序限制，因为一些行为可以不同的顺序进行和/或与其它行为或事件同时进行。此外，并非所有说明性的行为或事件对实施本发明的方法都为必需的。Several aspects of the invention are described below with reference to example applications for illustration. It should be understood that numerous specific details, relationships, and methods are set forth to provide a thorough understanding of the invention. One of ordinary skill in the relevant art will readily recognize, however, that the invention may be practiced without one or more of the specific details or may be practiced otherwise. The invention is not limited by the ordering of acts or events, as some acts may occur in different orders and/or concurrently with other acts or events. Moreover, not all illustrated acts or events are required to practice the methods of the invention.
本文公开的所有基因、基因名称和基因产物意图对应来自任何物种的同源物，对该物种而言本文公开的组合物和方法为适用的。因此，该术语包括但不限于来自人和小鼠的基因和基因产物。理解的是，当公开来自具体物种的基因或基因产物时，意图此公开仅为示范性的，并且除非其出现的上下文中明确指示，否则不应理解为限制。因此，例如，对于本文公开的在一些实施方案中有关哺乳动物核酸和氨基酸序列的基因而言，意图包括来自其它动物(包括但不限于其它哺乳动物、鱼类、两栖动物、爬行动物和鸟类)的同源和/或直向同源基因和基因产物。在实施方案中，所述基因或核酸序列为人的。All genes, gene names and gene products disclosed herein are intended to correspond to homologues from any species for which the compositions and methods disclosed herein are applicable. Thus, the term includes, but is not limited to, genes and gene products from humans and mice. It is understood that when a gene or gene product from a particular species is disclosed, it is intended that such disclosure is exemplary only, and should not be taken as limiting unless clearly indicated by the context in which it appears. Thus, for example, for genes disclosed herein which in some embodiments relate to mammalian nucleic acid and amino acid sequences, it is intended to include genes from other animals including, but not limited to, other mammals, fish, amphibians, reptiles, and birds. ) homologous and/or orthologous genes and gene products. In embodiments, the gene or nucleic acid sequence is human.
定义definition
本文所用的术语仅以描述具体的实施方案为目的而不意图限制本发明。除非上下文另有明确指示，否则本文所用的单数形式“一”、和“所述”也意图包括复数形式。此外，就术语“包括的”、“包括”、“具有的”、“具有”、“含有”或其变型在详述和/或权利要求中所用的程度而言，这类术语意图以类似于术语“包含”的方式是包括在内的。The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the singular forms "a", "an", and "the" are intended to include the plural forms as well, unless the context clearly dictates otherwise. Furthermore, to the extent the terms "comprising", "comprising", "having", "having", "containing" or variations thereof are used in the detailed description and/or claims, such terms are intended to be used in a manner similar to The term "comprising" means inclusive.
术语“约”或“大约”意为在由本领域普通技术人员所确定的具体值的可接受误差范围之内，这部分取决于该值是如何测定或确定的，即，测量系统的限制。例如，按照本领域的实践，“约”可意为在1之内或大于1的标准差。或者，“约”可意为最高达给定值的20％，优选10％，更优选5％，和还更优选1％的范围。或者，具体地关于生物系统或过程，该术语可意为在值的一定数量级之内，优选在值的5倍之内，更优选在2倍之内。当本申请和权利要求描述具体值时，除非另作说明，否则应假设术语“约”意为在具体值的可接受误差范围之内。The term "about" or "approximately" means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, depending in part on how the value was measured or determined, ie, the limitations of the measurement system. For example, "about" can mean within 1 or greater than 1 standard deviation, per the practice in the art. Alternatively, "about" can mean a range of up to 20%, preferably 10%, more preferably 5%, and still more preferably 1% of a given value. Alternatively, with particular reference to biological systems or processes, the term may mean within a certain order of magnitude, preferably within 5 times, more preferably within 2 times, the value. When this application and claims describe specific values, unless otherwise stated, the term "about" means within an acceptable error range for the specific value, it should be assumed.
本文所用的术语“mRNA”意为目前已知的靶向基因的mRNA转录物，以及任何可阐明的其它转录物。The term "mRNA" as used herein means the currently known mRNA transcript of a targeted gene, as well as any other transcripts that can be elucidated.
“反义寡核苷酸”或“反义化合物”意为与另一个RNA或DNA(靶RNA、DNA)结合的RNA或DNA分子。例如，如果其为RNA寡核苷酸，则其通过RNA-RNA相互作用结合另一个RNA靶标并改变靶RNA的活性。反义寡核苷酸可增量调节或减量调节特定多核苷酸的表达和/或功能。该定义意在包括从治疗、诊断或其它观点来看有用的任何外源RNA或DNA分子。这类分子包括例如反义RNA或DNA分子、干扰RNA(RNAi)、微小RNA、诱饵RNA分子、siRNA、酶促RNA、治疗用编辑RNA(therapeutic editing RNA)以及激动剂和拮抗剂RNA、反义寡聚化合物、反义寡核苷酸、外部指导序列(EGS)寡核苷酸、可变剪接物(alternate splicer)、引物、探针以及其它与靶核酸的至少一部分杂交的寡聚化合物。因此，可将这些化合物以单链、双链、部分单链或环状寡聚化合物的形式引入。"Antisense oligonucleotide" or "antisense compound" means an RNA or DNA molecule that binds to another RNA or DNA (target RNA, DNA). For example, if it is an RNA oligonucleotide, it binds another RNA target through RNA-RNA interactions and alters the activity of the target RNA. Antisense oligonucleotides can up-regulate or down-regulate the expression and/or function of a particular polynucleotide. This definition is intended to include any exogenous RNA or DNA molecule useful from a therapeutic, diagnostic or other standpoint. Such molecules include, for example, antisense RNA or DNA molecules, interfering RNA (RNAi), microRNA, decoy RNA molecules, siRNA, enzymatic RNA, therapeutic editing RNA, and agonist and antagonist RNA, antisense Oligomeric compounds, antisense oligonucleotides, external guide sequence (EGS) oligonucleotides, alternate splicers, primers, probes, and other oligomeric compounds that hybridize to at least a portion of a target nucleic acid. Thus, these compounds can be introduced as single-stranded, double-stranded, partially single-stranded or cyclic oligomeric compounds.
在本发明的上下文中，术语“寡核苷酸”是指核糖核酸(RNA)或脱氧核糖核酸(DNA)或其模拟物的寡聚物或聚合物。术语“寡核苷酸”，也包括天然和/或经修饰单体或键(linkage)的线性或环状寡聚体，包括脱氧核糖核苷、核糖核苷、其取代和α-异头物形式、肽核酸(PNA)、锁定核酸(LNA)、硫代磷酸酯、甲基膦酸酯等。寡核苷酸能够通过单体与单体相互作用的规律模式(例如沃森-克里克(Watson-Crick)型碱基配对、或反型碱基配对等)特异地结合靶多核苷酸。In the context of the present invention, the term "oligonucleotide" refers to an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or mimetics thereof. The term "oligonucleotide" also includes linear or cyclic oligomers of natural and/or modified monomers or linkages, including deoxyribonucleosides, ribonucleosides, substitutions thereof and α-anomers forms, Peptide Nucleic Acid (PNA), Locked Nucleic Acid (LNA), Phosphorothioate, Methylphosphonate, etc. Oligonucleotides are capable of regular patterns of monomer-monomer interaction (e.g. Watson-Crick type base pairing, or reverse type base pairing, etc.) to specifically bind the target polynucleotide.
寡核苷酸可为“嵌合的”，即，由不同的区组成。在本发明的上下文中，“嵌合的”化合物为寡核苷酸，其包含两个或更多个化学区，例如，DNA区、RNA区、PNA区等。每个化学区由至少一个单体单元(即，在寡核苷酸化合物的情况下为核苷酸)组成。这些寡核苷酸典型地包含至少一个区，其中所述寡核苷酸为经修饰的以表现出一种或更多种所需特性。寡核苷酸的所需特性包括但不限于，例如增强的对核酸酶降解的抗性、增强的细胞摄取和/或增强的对靶核酸的结合亲和力。因此寡核苷酸的不同区可具有不同的特性。本发明的嵌合寡核苷酸可形成为两种或更多种如上所述的寡核苷酸、修饰的寡核苷酸、寡聚核苷和/或寡核苷酸类似物的混合结构。Oligonucleotides may be "chimeric", ie, composed of distinct regions. In the context of the present invention, "chimeric" compounds are oligonucleotides, which comprise two or more chemical regions, eg, DNA regions, RNA regions, PNA regions, and the like. Each chemical domain consists of at least one monomeric unit (ie, a nucleotide in the case of an oligonucleotide compound). These oligonucleotides typically comprise at least one region wherein the oligonucleotide is modified to exhibit one or more desired properties. Desirable properties of oligonucleotides include, but are not limited to, for example, enhanced resistance to nuclease degradation, enhanced cellular uptake, and/or enhanced binding affinity for target nucleic acids. Different regions of an oligonucleotide may thus have different properties. Chimeric oligonucleotides of the invention may be formed as mixed structures of two or more oligonucleotides, modified oligonucleotides, oligonucleotides and/or oligonucleotide analogs as described above .
寡核苷酸可由可“全符合状态(in“register”)”地连接或通过间隔物连接的区组成，所述“全符合状态”地连接即此时单体像在天然DNA一样连续地连接。所述间隔物意在构成区之间的共价“桥”，并在一些情况下具有不超过约100个碳原子的长度。所述间隔物可携带不同的官能度，例如具有正或负电荷的，携带特殊的核酸结合特性(嵌入剂、沟结合剂、毒素、荧光团等)，为亲脂的、诱导特殊的二级结构如例如诱导α-螺旋的含丙氨酸的肽。Oligonucleotides may be composed of regions that may be linked "in "register", where the monomers are linked consecutively as in native DNA, or via spacers . The spacers are intended to constitute covalent "bridges" between regions, and in some cases have a length of no more than about 100 carbon atoms. The spacers can carry different functionalities, e.g. have positive or negative charges, carry specific nucleic acid binding properties (intercalators, groove binders, toxins, fluorophores, etc.), be lipophilic, induce specific secondary Structures such as, for example, alanine-containing peptides that induce α-helices.
本文所用的“抗病毒基因”包括所有家族成员、突变体、等位基因、片段、物种、编码序列和非编码序列、有义和反义多核苷酸链等。"Antiviral gene" as used herein includes all family members, mutants, alleles, fragments, species, coding and non-coding sequences, sense and antisense polynucleotide strands, and the like.
本文所用的词语RIG1、RIG-1、RIG-I、DEAD盒蛋白58、DKFZp434J1111、DKFZp686N19181、FLJ13599、视黄酸可诱导的基因1蛋白、视黄酸可诱导的基因I蛋白被认为与文献中相同，并在本申请中可互换地使用。The terms RIG1, RIG-1, RIG-I, DEAD box protein 58, DKFZp434J1111, DKFZp686N19181, FLJ13599, retinoic acid inducible gene 1 protein, retinoic acid inducible gene 1 protein as used herein are considered to be the same as in the literature , and are used interchangeably in this application.
本文所用的词语MDA5、MDA-5、黑素瘤分化相关的蛋白5、Helicard、含有2个CARD结构域的解螺旋酶、Hlcd、IDDM19、含有干扰素诱导的解螺旋酶C结构域的蛋白1、用解螺旋酶C结构域蛋白1诱导的干扰素、MGC133047、莫拉丁酯减量调节的蛋白、RH116、RNA解螺旋酶-DEAD盒蛋白116被认为与文献中相同，并在本申请中可互换地使用。As used herein, the terms MDA5, MDA-5, melanoma differentiation-associated protein 5, Helicard, helicase containing 2 CARD domains, Hlcd, IDDM19, interferon-inducible helicase C-domain containing protein 1 , Interferon induced with helicase C domain protein 1, MGC133047, Moradin ester down-regulated protein, RH116, RNA helicase-DEAD box protein 116 are considered the same as in the literature and can be used in this application used interchangeably.
本文所用的词语IFNA1、IFNAI1、G10P1、GARG-16、IFI56、IFI-56、IFI-56K、IFIT-1、干扰素诱导的56kDa蛋白、干扰素诱导的含有三十四肽重复序列1的蛋白、ISG56、RNM561被认为与文献中相同，并在本申请中可互换地使用。As used herein, the terms IFNA1, IFNAI1, G10P1, GARG-16, IFI56, IFI-56, IFI-56K, IFIT-1, interferon-induced 56 kDa protein, interferon-induced tetratetradetrapeptide repeat 1-containing protein, ISG56, RNM561 are considered the same as in the literature and are used interchangeably in this application.
本文所用的术语“对……特异性的寡核苷酸”或“靶向……的寡核苷酸”是指具有以下序列的寡核苷酸：该序列(i)能够与被靶向的基因的一部分形成稳定的复合体，或(ii)能够与被靶向的基因的mRNA转录物的一部分形成稳定的双链体。复合体和双链体的稳定性可通过理论计算和/或体外测定来确定。用于确定杂交复合体和双链体的稳定性的示例性测定法描述于下文实施例中。As used herein, the term "oligonucleotide specific for ..." or "oligonucleotide targeting ..." refers to an oligonucleotide having a sequence (i) capable of interacting with the targeted A portion of the gene forms a stable complex, or (ii) is capable of forming a stable duplex with a portion of the mRNA transcript of the gene being targeted. Complex and duplex stability can be determined by theoretical calculations and/or in vitro assays. Exemplary assays for determining the stability of hybridization complexes and duplexes are described in the Examples below.
本文所用的术语“靶核酸”包括DNA、从这类DNA转录的RNA(包括前mRNA和mRNA)，以及从这类RNA衍生的cDNA、编码序列、非编码序列、有义或反义多核苷酸。寡聚化合物与其靶核酸的特异性杂交干扰核酸的正常功能。这种通过特异地与靶核酸杂交的化合物对该靶核酸功能的调节，一般称为“反义”。待干扰的DNA功能包括例如复制和转录。待干扰的RNA功能，包括所有的生活机能，例如，RNA向蛋白质翻译位点的易位、蛋白质自RNA的翻译、产生一种或更多种mRNA种类的RNA剪接，以及可由RNA参与或促进的催化活性。对靶核酸功能的这类干扰的整体效果为对编码产物或寡核苷酸表达的调节。As used herein, the term "target nucleic acid" includes DNA, RNA (including pre-mRNA and mRNA) transcribed from such DNA, and cDNA, coding sequences, non-coding sequences, sense or antisense polynucleotides derived from such RNA . Specific hybridization of oligomeric compounds to their target nucleic acid interferes with the normal function of the nucleic acid. Such regulation of the function of a target nucleic acid by a compound that specifically hybridizes to the target nucleic acid is generally referred to as "antisense". DNA functions to be interfered with include, for example, replication and transcription. RNA function to be interfered with, including all vital functions such as translocation of RNA to protein translation sites, translation of proteins from RNA, RNA splicing to produce one or more mRNA species, and processes that may be engaged or facilitated by RNA catalytic activity. The overall effect of such interference with target nucleic acid function is modulation of expression of the encoded product or oligonucleotide.
RNA干扰“RNAi”由双链RNA(dsRNA)分子介导，该分子具有与其“靶”核酸序列的序列特异的同源性。在本发明的某些实施方案中，介体为5-25个核苷酸的“小干扰”RNA双链体(siRNA)。siRNA源自称为切酶(Dicer)的RNA酶对dsRNA的加工。siRNA双链体产物被募集到叫做RISC(RNA诱导的沉默复合体)的多蛋白siRNA复合体中。不希望受任何具体理论所约束，认为随后RISC被引向靶核酸(适当地为mRNA)，其中siRNA双链体以序列特异性的方式相互作用来介导催化方式的切割。可根据本发明使用的小干扰RNA，可根据本领域众所周知和普通技术人员熟悉的程序来合成和使用。用于本发明的方法中的小干扰RNA适当地包含约1-约50个核苷酸(nt)。在非限制性实施方案的实例中，siRNA可包含约5-约40个核苷酸、约5-约30个核苷酸、约10-约30个核苷酸、约15-约25个核苷酸或约20-25个核苷酸。RNA interference "RNAi" is mediated by double-stranded RNA (dsRNA) molecules that have sequence-specific homology to their "target" nucleic acid sequences. In certain embodiments of the invention, the mediator is a "small interfering" RNA duplex (siRNA) of 5-25 nucleotides. siRNA is derived from the processing of dsRNA by an RNase called Dicer. The siRNA duplex product is recruited into a multiprotein siRNA complex called RISC (RNA-induced silencing complex). Without wishing to be bound by any particular theory, it is believed that RISC is then directed to the target nucleic acid, suitably mRNA, where the siRNA duplex interacts in a sequence-specific manner to mediate cleavage in a catalytic manner. Small interfering RNAs that can be used in accordance with the present invention can be synthesized and used according to procedures well known in the art and familiar to those of ordinary skill. Small interfering RNAs for use in the methods of the invention suitably comprise from about 1 to about 50 nucleotides (nt). In examples of non-limiting embodiments, siRNAs may comprise about 5 to about 40 nucleotides, about 5 to about 30 nucleotides, about 10 to about 30 nucleotides, about 15 to about 25 cores nucleotides or about 20-25 nucleotides.
适当寡核苷酸的挑选通过使用电脑程序来促进，该程序自动比对核酸序列并指出具有同一性或同源性的区。将这类程序用于比较通过例如搜索诸如GenBank等的数据库或通过测序PCR产物而获得的核酸序列。对来自一系列物种的核酸序列的比较，允许选择在物种之间显示适度同一性的核酸序列。在未测序基因的情况下，进行DNA印迹来确定在靶物种和其它物种的基因之间的同一性程度。如本领域众所周知的，通过在不同的严格程度下进行DNA印迹，可能获得同一性的近似衡量。这些程序允许选择以下寡核苷酸，其对待控制的受试者中的靶核酸序列表现出高度的互补性，并且对其它物种中的相应核酸序列表现出较低程度的互补性。本领域的技术人员将认识到，在挑选用于本发明的适当的基因区方面具有相当大的自由。Selection of appropriate oligonucleotides is facilitated by the use of computer programs that automatically align nucleic acid sequences and indicate regions of identity or homology. Such programs are used to compare nucleic acid sequences obtained by, for example, searching databases such as GenBank or by sequencing PCR products. Comparison of nucleic acid sequences from a range of species allows the selection of nucleic acid sequences that show modest identities between species. In the case of unsequenced genes, Southern blots are performed to determine the degree of identity between genes of the target species and other species. It is possible to obtain approximate measures of identity by performing Southern blots at varying degrees of stringency, as is well known in the art. These procedures allow the selection of oligonucleotides that exhibit a high degree of complementarity to a target nucleic acid sequence in the subject to be controlled and a lower degree of complementarity to corresponding nucleic acid sequences in other species. Those skilled in the art will recognize that there is considerable freedom in selecting appropriate gene regions for use in the present invention.
“酶促RNA”意为具有酶促活性的RNA分子。酶促核酸(核酶)通过首先结合靶RNA来起作用。这类结合通过酶促核酸的靶结合部分进行，所述靶结合部分保持紧密靠近进行切割靶RNA的分子的酶促部分。因此，酶促核酸首先识别而后通过碱基配对结合靶RNA，且一旦结合正确的位点，即进行酶促切割靶RNA。"Enzymatic RNA" means an RNA molecule having enzymatic activity. Enzymatic nucleic acids (ribozymes) work by first binding to a target RNA. Such binding occurs through the target binding portion of the enzymatic nucleic acid, which is held in close proximity to the enzymatic portion of the molecule that cleaves the target RNA. Thus, the enzymatic nucleic acid first recognizes and then binds the target RNA through base pairing, and once bound to the correct site, enzymatically cleaves the target RNA.
“诱饵RNA”意为模拟配体的天然结合域的RNA分子。因此诱饵RNA与天然结合靶标竞争与特异性配体的结合。例如，已显示HIV反式激活应答(TAR)RNA的过表达可充当“诱饵”并有效地结合HIV tat蛋白，从而阻止其结合在HIV RNA中编码的TAR序列。这意指特定实例。本领域的技术人员将认识到，这只是一个实例，而其它的实施方案可使用本领域一般已知的技术容易地产生。"Decoy RNA" means an RNA molecule that mimics the natural binding domain of a ligand. The decoy RNA thus competes with the natural binding target for binding to the specific ligand. For example, overexpression of HIV transactivation response (TAR) RNA has been shown to act as a "decoy" and efficiently bind the HIV tat protein, thereby preventing it from binding the TAR sequence encoded in HIV RNA. This means a specific instance. Those skilled in the art will recognize that this is only one example and that other embodiments can be readily generated using techniques generally known in the art.
本文所用的术语“单体”通常指以下单体，其通过磷酸二酯键或其类似物连接以形成大小范围从几个单体单元(例如从约3-4)到约数百个单体单元的寡核苷酸。磷酸二酯键的类似物包括：硫代磷酸酯、二硫代磷酸酯、甲基膦酸酯、硒代磷酸酯、氨基磷酸酯等，如下文更充分地描述的。As used herein, the term "monomer" generally refers to monomers linked by phosphodiester linkages or analogs thereof to form monomers ranging in size from a few monomer units (e.g., from about 3-4) to about several hundred monomer units. units of oligonucleotides. Analogs of a phosphodiester linkage include: phosphorothioate, phosphorodithioate, methylphosphonate, phosphoroselenoate, phosphoramidate, and the like, as described more fully below.
术语“核苷酸”涵盖天然存在的核苷酸和非天然存在的核苷酸。本领域的技术人员应清楚的是，先前认为“非天然存在”的多种核苷酸后来已在自然中发现。因此，“核苷酸”不仅包括已知的含嘌呤和嘧啶杂环的分子，而且还包括其杂环类似物和互变异构体。其它类型的核苷酸的说明性实例为以下分子，其含有腺嘌呤、鸟嘌呤、胸腺嘧啶、胞嘧啶、尿嘧啶、嘌呤、黄嘌呤、二氨基嘌呤、8-氧代-N6-甲基腺嘌呤、7-脱氮黄嘌呤、7-脱氮鸟嘌呤、N4，N4-桥亚乙基胞嘧啶(ethanocytosin)、N6，N6-桥亚乙基-2，6-二氨基嘌呤、5-甲基胞嘧啶、5-(C3-C6)-炔基胞嘧啶、5-氟尿嘧啶、5-溴尿嘧啶、假异胞嘧啶、2-羟基-5-甲基-4-三唑并吡啶、异胞嘧啶、异鸟嘌呤、肌苷和在美国专利第5,432,272号中描述的“非天然存在的”核苷酸。术语“核苷酸”意在涵盖这些实例以及其类似物和互变异构体中的每一个和全部。尤其令人关注的核苷酸为含腺嘌呤、鸟嘌呤、胸腺嘧啶、胞嘧啶和尿嘧啶的核苷酸，其被认为是有关人中的治疗和诊断应用的天然存在核苷酸。核苷酸包括天然2’-脱氧和2’-羟基糖，例如，如Kornberg和Baker，DNA复制(DNA Replication)，第2版(Freeman，San Francisco，1992)中所述的以及其类似物。The term "nucleotide" encompasses both naturally occurring and non-naturally occurring nucleotides. It will be apparent to those skilled in the art that a variety of nucleotides previously considered "non-naturally occurring" have subsequently been found in nature. Thus, "nucleotide" includes not only known molecules containing purine and pyrimidine heterocycles, but also heterocycle analogs and tautomers thereof. Illustrative examples of other types of nucleotides are molecules containing adenine, guanine, thymine, cytosine, uracil, purine, xanthine, diaminopurine, 8-oxo-N6-methyladenine Purine, 7-deazaxanthine, 7-deazaguanine, N4, N4-ethanocytosin, N6, N6-ethanide-2,6-diaminopurine, 5-methyl Cytosine, 5-(C3-C6)-alkynylcytosine, 5-fluorouracil, 5-bromouracil, pseudoisocytosine, 2-hydroxy-5-methyl-4-triazolopyridine, isocytosine Pyrimidine, isoguanine, inosine, and "non-naturally occurring" nucleotides described in US Patent No. 5,432,272. The term "nucleotide" is intended to cover each and all of these examples, as well as analogs and tautomers thereof. Nucleotides of particular interest are those containing adenine, guanine, thymine, cytosine and uracil, which are considered naturally occurring nucleotides for therapeutic and diagnostic applications in humans. Nucleotides include natural 2'-deoxy and 2'-hydroxy sugars, e.g., as described in Kornberg and Baker, DNA Replication, 2nd Ed. (Freeman, San Francisco, 1992) and analogs thereof.
提及核苷酸的“类似物”包括具有经修饰的碱基部分和/或经修饰的糖部分的合成核苷酸。这类类似物包括设计以增强结合特性的合成核苷酸，所述结合特性为例如双链体或三链体稳定性、特异性等。Reference to "analogues" of nucleotides includes synthetic nucleotides having modified base moieties and/or modified sugar moieties. Such analogs include synthetic nucleotides designed to enhance binding properties such as duplex or triplex stability, specificity, and the like.
本文所用的“杂交”意为寡聚化合物的基本上互补链的配对。一种配对的机理涉及寡聚化合物的链的互补核苷或核苷酸碱基(核苷酸)之间的氢键合，其可为沃森-克里克、或反氢键合。例如，腺嘌呤和胸腺嘧啶为互补的核苷酸，其通过形成氢键配对。杂交可在各种环境下发生。As used herein, "hybridization" means the pairing of substantially complementary strands of oligomeric compounds. One mechanism of pairing involves hydrogen bonding between complementary nucleoside or nucleotide bases (nucleotides) of strands of oligomeric compounds, which can be Watson-Crick, or reverse hydrogen bonding. For example, adenine and thymine are complementary nucleotides that pair by forming hydrogen bonds. Hybridization can occur under a variety of circumstances.
反义化合物为“可特异地杂交的”，如果所述化合物与靶核酸的结合干扰靶核酸的正常功能而导致功能和/或活性的调节，并且在需要特异性结合的条件下存在足够程度的互补性来避免所述反义化合物与非靶核酸序列的非特异性结合，所述条件即在体内测定或治疗性处理情况中的生理条件下，以及其中在体外测定情况下进行测定的条件下。An antisense compound is "specifically hybridizable" if the binding of the compound to the target nucleic acid interferes with the normal function of the target nucleic acid resulting in modulation of function and/or activity, and a sufficient degree is present under conditions requiring specific binding. Complementarity to avoid non-specific binding of the antisense compound to non-target nucleic acid sequences, ie under physiological conditions in the case of in vivo assays or therapeutic treatments, and under conditions in which the assay is performed in the case of in vitro assays.
本文所用的短语“严格杂交条件”或“严格条件”是指以下条件，在该条件下本发明的化合物与其靶序列杂交，但与最少数量的其它序列杂交。严格条件为序列依赖的且在不同环境下将不同，在本发明的上下文中，在其下寡聚化合物与靶序列杂交的“严格条件”由寡聚化合物的性质和组成以及正在其中研究它们的试验来确定。一般而言，严格杂交条件包括低浓度(＜0.15M)的含有诸如Na++或K++等无机阳离子的盐(即，低离子强度)、温度高于20℃-25℃、低于寡聚化合物：靶序列复合体的Tm，以及存在变性剂，例如甲酰胺、二甲基甲酰胺、二甲基亚砜，或去污剂十二烷基硫酸钠(SDS)。例如，杂交率对于每1％甲酰胺减少1.1％。高严格杂交条件的实例为0.1X氯化钠-柠檬酸钠缓冲液(SSC)/0.1％(w/v)SDS、60℃下达30分钟。As used herein, the phrase "stringent hybridization conditions" or "stringent conditions" refers to conditions under which a compound of the invention will hybridize to its target sequence, but to a minimal number of other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances, and in the context of the present invention, "stringent conditions" under which oligomeric compounds hybridize to target sequences consist of the nature and composition of the oligomeric compounds and the conditions in which they are being studied. Test to be sure. In general, stringent hybridization conditions include low concentrations (<0.15M) of salts containing inorganic cations such as Na++ or K++ (i.e., low ionic strength), temperatures above 20°C-25°C, temperatures below oligomeric compound: target The Tm of the sequence complex, and the presence of denaturing agents such as formamide, dimethylformamide, dimethyl sulfoxide, or the detergent sodium dodecyl sulfate (SDS). For example, the hybridization rate decreases by 1.1% for every 1% of formamide. An example of highly stringent hybridization conditions is 0.1X sodium chloride-sodium citrate buffer (SSC)/0.1% (w/v) SDS at 60°C for 30 minutes.
本文所用的“互补的”是指在一条或两条寡聚链上两个核苷酸之间精确配对的能力。例如，如果在反义化合物的某个位置上的核碱基能够与在靶核酸的某个位置上的核碱基氢键合，所述靶核酸为DNA、RNA或寡核苷酸分子，则认为所述寡核苷酸和所述靶核酸之间氢键合的位置为互补位置。当可彼此氢键合的核苷酸占据了每个分子中足够数量的互补位置时，寡聚化合物和另外的DNA、RNA或寡核苷酸分子为彼此互补的。因此，“可特异性杂交的”和“互补的”为以下术语，其用于表示在足够数量的核苷酸上有足够程度的精确配对或互补性，使得稳定和特异的结合发生在寡聚化合物和靶核酸之间。As used herein, "complementary" refers to the ability to precisely pair between two nucleotides on one or both oligomeric strands. For example, if a nucleobase at a certain position in an antisense compound is capable of hydrogen bonding to a nucleobase at a certain position in a target nucleic acid, which is a DNA, RNA or oligonucleotide molecule, then The location of hydrogen bonding between the oligonucleotide and the target nucleic acid is considered to be the location of complementarity. An oligomeric compound and an additional DNA, RNA or oligonucleotide molecule are complementary to each other when nucleotides capable of hydrogen bonding to each other occupy a sufficient number of complementary positions in each molecule. Accordingly, "specifically hybridizable" and "complementary" are terms used to denote a sufficient degree of precise pairing or complementarity over a sufficient number of nucleotides such that stable and specific binding occurs at the oligomeric between the compound and the target nucleic acid.
在本领域理解，寡聚化合物的序列不需要与其可特异地杂交的靶核酸的序列100％互补。此外，寡核苷酸可在一个或更多个区段上杂交，使得间插或邻近的区段不涉及杂交事件(例如，环结构、错配或发夹结构)。本发明的寡聚化合物包含与其靶向的靶核酸序列内的靶区至少约70％、或至少约75％、或至少约80％、或至少约85％、或至少约90％、或至少约95％、或至少约99％的序列互补性。例如，其中反义化合物的20个核苷酸中有18个与靶区互补且因而会特异地杂交的反义化合物，表示90％互补性。在此实例中，余下的非互补核苷酸可与互补核苷酸是聚簇或散布的且不需要彼此邻接或邻接互补核苷酸。因此，长度为18个核苷酸的反义化合物具有4(四)个非互补核苷酸，该非互补核苷酸位于与靶核酸完全互补的两个区的侧翼，所述反义化合物会具有与靶核酸77.8％的总互补性，因此落入本发明的范围内。反义化合物与靶核酸区的互补性百分比可使用本领域已知的BLAST程序(基本局部比对搜索工具)和PowerBLAST程序常规地确定。同源性、序列同一性或互补性百分比可通过例如Gap程序(Wisconsin序列分析包，Unix操作系统版本8，Genetics ComputerGroup，University Research Park，Madison Wis.)使用默认设置来确定，该程序使用Smith和Waterman的算法(Adv.Appl.Math.，(1981)2，482-489)。It is understood in the art that the sequence of an oligomeric compound need not be 100% complementary to the sequence of its target nucleic acid to which it is specifically hybridizable. In addition, oligonucleotides can hybridize over one or more segments such that intervening or adjacent segments are not involved in hybridization events (eg, loop structures, mismatches, or hairpin structures). The oligomeric compounds of the present invention comprise at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 99%, sequence complementarity. For example, an antisense compound in which 18 of the 20 nucleotides of the antisense compound are complementary to the target region and thus specifically hybridize, represents 90% complementarity. In this example, the remaining non-complementary nucleotides may be clustered or interspersed with the complementary nucleotides and need not be adjacent to each other or to the complementary nucleotides. Thus, an antisense compound that is 18 nucleotides in length has 4 (four) non-complementary nucleotides flanking two regions that are fully complementary to the target nucleic acid, said antisense compound would Has a total complementarity of 77.8% to the target nucleic acid and thus falls within the scope of the present invention. The percent complementarity of an antisense compound to a region of a target nucleic acid can be routinely determined using the BLAST programs (Basic Local Alignment Search Tool) and the PowerBLAST programs known in the art. Homology, sequence identity or percent complementarity can be determined using default settings by, for example, the Gap program (Wisconsin Sequence Analysis Package, Unix Operating System Version 8, Genetics Computer Group, University Research Park, Madison Wis.), which uses Smith and Waterman's algorithm (Adv. Appl. Math., (1981) 2, 482-489).
本文所用的术语“解链温度(Tm)”是指以下温度，在限定的离子强度、pH和核酸浓度下，在该温度下平衡时50％与靶序列互补的寡核苷酸与靶序列杂交。典型地，对于短寡核苷酸(例如，10-50个核苷酸)而言严格条件为以下条件，其中盐浓度至少为约0.01-1.0M Na离子浓度(或其它盐)，pH 7.0-8.3且温度至少为约30℃。严格条件也可通过外加诸如甲酰胺等去稳定剂来达到。As used herein, the term "melting temperature (Tm)" refers to the temperature at which 50% of the oligonucleotides complementary to the target sequence hybridize to the target sequence at a defined ionic strength, pH and nucleic acid concentration . Typically, stringent conditions for short oligonucleotides (e.g., 10-50 nucleotides) are those in which the salt concentration is at least about 0.01-1.0 M Na ion concentration (or other salts), pH 7.0- 8.3 and a temperature of at least about 30°C. Stringent conditions can also be achieved by adding destabilizing agents such as formamide.
本文所用的“调节”意为在基因表达方面的增加(刺激)或减少(抑制)。As used herein, "modulation" means an increase (stimulation) or decrease (inhibition) in gene expression.
术语“变体”，当用于多核苷酸序列的情况下时，可包括有关野生型基因的多核苷酸序列。此定义也可包括，例如，“等位基因的”、“剪接”、“物种”或“多态的”变体。剪接变体可具有与参比分子显著的同一性，但因为在mRNA加工期间外显子的可变剪接而通常具有更多或更少数量的多核苷酸。对应的多肽可具有附加的功能域或不存在域。物种变体为在不同物种之间不同的多核苷酸序列。本发明中尤其实用的是野生型基因产物的变体。变体可由核酸序列中的至少一个突变产生并可导致产生改变的mRNA或者其结构或功能可能改变或不变的多肽。任何给定的天然或重组基因可不具有、具有一个或许多等位基因形式。产生变体的常见突变变化一般归因于核苷酸的自然缺失、添加或取代。这些变化类型中的每一个可单独或与其它类型联合发生，在给定序列中发生一次或更多次。The term "variant", when used in the context of polynucleotide sequences, may include polynucleotide sequences related to wild-type genes. This definition may also include, for example, "allelic", "splice", "species" or "polymorphic" variants. A splice variant may have significant identity to a reference molecule, but typically have a greater or lesser number of polynucleotides due to alternative splicing of exons during mRNA processing. Corresponding polypeptides may have additional functional domains or be absent. Species variants are polynucleotide sequences that differ between different species. Of particular utility in the present invention are variants of the wild-type gene product. A variant may result from at least one mutation in the nucleic acid sequence and may result in an altered mRNA or a polypeptide whose structure or function may or may not be altered. Any given native or recombinant gene may have none, one or many allelic forms. Common mutational changes that produce variants are generally due to natural deletions, additions, or substitutions of nucleotides. Each of these types of changes may occur alone or in combination with the others, one or more times in a given sequence.
产生的多肽一般将具有相对于彼此的显著的氨基酸同一性。多态变体为在给定物种的个体之间特定基因的多核苷酸序列的变化。多态变体也可包括“单核苷酸多态性”(SNP)或单碱基突变，其中多核苷酸序列因一个碱基而不同。SNP的存在可指示例如具有疾病状态倾向(即与抗性相对的易感性)的某个种群。The resulting polypeptides will generally have significant amino acid identity relative to each other. A polymorphic variant is a variation in the polynucleotide sequence of a particular gene between individuals of a given species. Polymorphic variants may also include "single nucleotide polymorphisms" (SNPs), or single base mutations, in which the sequence of a polynucleotide differs by one base. The presence of a SNP can indicate, for example, a certain population with a predisposition to a disease state (ie, susceptibility as opposed to resistance).
衍生多核苷酸包括经过化学修饰的核酸，例如用烷基、酰基或氨基置换氢。衍生物(例如，衍生寡核苷酸)可包含非天然存在的部分，例如改变的糖部分或糖间键。这些中示例性的是硫代磷酸酯及本领域已知的其它含硫的种类。衍生核酸也可含有标记，包括放射性核苷酸、酶、荧光剂、化学发光剂、显色剂、底物、辅因子、抑制剂、磁性颗粒，等等。Derivatized polynucleotides include nucleic acids that have been chemically modified, eg, by replacing a hydrogen with an alkyl, acyl or amino group. Derivatives (eg, derivatized oligonucleotides) may contain non-naturally occurring moieties, such as altered sugar moieties or intersugar linkages. Illustrative of these are phosphorothioates and other sulfur-containing species known in the art. Derivatized nucleic acids may also contain labels, including radionucleotides, enzymes, fluorescent agents, chemiluminescent agents, chromogenic agents, substrates, cofactors, inhibitors, magnetic particles, and the like.
“衍生的”多肽或肽为经修饰的多肽或肽，例如，通过糖基化、聚乙二醇化、磷酸化作用、硫酸盐化作用、还原/烷基化、酰化、化学偶联或温性福尔马林处理。也可将衍生物修饰以含有可检测标记(直接地或间接地)，包括但不限于放射性同位素、荧光和酶标记。A "derivatized" polypeptide or peptide is one that has been modified, e.g., by glycosylation, pegylation, phosphorylation, sulfation, reduction/alkylation, acylation, chemical conjugation, or mild stimulation. Malmarin handles. Derivatives may also be modified to contain detectable labels (directly or indirectly), including, but not limited to, radioisotopic, fluorescent, and enzymatic labels.
本文所用的术语“动物”或“患者”意在包括例如人、棉羊、麋鹿、鹿、长耳鹿、貂、哺乳动物、猴、马、牛、猪、山羊、狗、猫、大鼠、小鼠、鸟、鸡、爬行动物、鱼、昆虫和蜘蛛类。The term "animal" or "patient" as used herein is intended to include, for example, humans, sheep, elk, deer, mule deer, mink, mammals, monkeys, horses, cows, pigs, goats, dogs, cats, rats, Mice, birds, chickens, reptiles, fish, insects and arachnids.
“哺乳动物”涵盖通常在医疗护理下的温血哺乳动物(例如，人和驯养动物)。实例包括猫科动物、犬、马、牛科动物和人，以及仅仅人。"Mammal" encompasses warm-blooded mammals (eg, humans and domesticated animals) usually under medical care. Examples include feline, canine, equine, bovine, and human, as well as merely human.
“处理”或“治疗”涵盖对哺乳动物中疾病状态的治疗，并包括：(a)防止疾病状态出现于哺乳动物中，特别是当这类哺乳动物倾向于疾病状态但尚未诊断为患有该疾病状态时；(b)抑制疾病状态，例如，阻止其发展；和/或(c)减轻疾病状态，例如，引起疾病状态的退行直到达到所需的终末点。治疗也包括改善疾病的症状(例如，减少疼痛或不适)，其中这类改善可直接或可非直接地影响疾病(例如，原因、传递、表达等)。"Treatment" or "treatment" encompasses the treatment of a disease state in a mammal and includes: (a) preventing the disease state from occurring in the mammal, especially when such mammals are predisposed to the disease state but have not been diagnosed with the disease (b) inhibiting the disease state, e.g., arresting its development; and/or (c) ameliorating the disease state, e.g., causing regression of the disease state until a desired endpoint is reached. Treatment also includes ameliorating a symptom of a disease (eg, reducing pain or discomfort), where such amelioration may directly or indirectly affect the disease (eg, cause, transmission, expression, etc.).
本文所用的“癌症”是指在哺乳动物中发现的所有类型的癌症或新生物或恶性肿瘤，包括但不限于：白血病、淋巴瘤、黑素瘤、癌和肉瘤。癌症自身表现为包含癌症恶性细胞的“肿瘤”或组织。肿瘤的实例包括肉瘤和癌，例如但不限于：纤维肉瘤、粘液肉瘤、脂肪肉瘤、软骨肉瘤、骨肉瘤、脊索瘤、血管肉瘤、内皮肉瘤、淋巴管肉瘤、淋巴管内皮肉瘤、滑膜瘤、间皮瘤、尤因氏瘤(Ewing′s tumor)、平滑肌肉瘤、横纹肌肉瘤、结肠癌、胰腺癌、乳腺癌、卵巢癌、前列腺癌、鳞状细胞癌、基底细胞癌、腺癌、汗腺癌、皮脂腺癌、乳头状癌、乳头状腺癌、囊腺癌、髓样癌、支气管癌、肾细胞癌、肝细胞瘤、胆管癌、绒毛膜癌、精原细胞瘤、胚胎性癌、维尔姆斯氏肿瘤(Wilms′tumor)、子宫颈癌、睾丸肿瘤、肺癌、小细胞肺癌、膀胱癌、上皮癌、神经胶质瘤、星形细胞瘤、髓母细胞瘤、颅咽管瘤、室管膜瘤、松果体瘤、成血管细胞瘤、听神经瘤、少突神经胶质瘤、脑膜瘤、黑素瘤、成神经细胞瘤和视网膜母细胞瘤。可通过根据本发明的公开组合物治疗的其它癌症，包括但不限于，例如，何杰金病、非何杰金淋巴瘤、多发性骨髓瘤、成神经细胞瘤、乳腺癌、卵巢癌、肺癌、横纹肌肉瘤、原发性血小板增多症、原发性巨球蛋白血症、小细胞肺肿瘤、原发性脑肿瘤、胃癌、结肠癌、恶性胰腺胰岛素瘤(insulanoma)、恶性类癌、尿道膀胱癌、恶化前皮肤病损、睾丸癌、淋巴瘤、甲状腺癌、成神经细胞瘤、食管癌、生殖泌尿道癌、恶性高钙血症、子宫颈癌、子宫内膜癌、肾上腺皮质癌和前列腺癌。"Cancer" as used herein refers to all types of cancers or neoplasms or malignancies found in mammals, including but not limited to: leukemias, lymphomas, melanomas, carcinomas and sarcomas. Cancer manifests itself as a "tumor" or tissue containing cancerous malignant cells. Examples of tumors include sarcomas and carcinomas such as, but not limited to: fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, endothelial sarcoma, lymphangiosarcoma, lymphangioendothelial sarcoma, synovoma, Mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma , sebaceous carcinoma, papillary carcinoma, papillary carcinoma, cystadenocarcinoma, medullary carcinoma, bronchial carcinoma, renal cell carcinoma, hepatoma, cholangiocarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm Wilms' tumor, cervical cancer, testicular tumor, lung cancer, small cell lung cancer, bladder cancer, epithelial cancer, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ventricular angioma melanoma, pineal tumor, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, and retinoblastoma. Other cancers treatable by the disclosed compositions according to the present invention include, but are not limited to, for example, Hodgkin's disease, non-Hodgkin's lymphoma, multiple myeloma, neuroblastoma, breast cancer, ovarian cancer, lung cancer , rhabdomyosarcoma, essential thrombocythemia, essential macroglobulinemia, small cell lung tumors, primary brain tumors, gastric cancer, colon cancer, malignant pancreatic insulinoma (insulanoma), malignant carcinoid, urethral bladder Cancer, premalignant skin lesions, testicular cancer, lymphoma, thyroid cancer, neuroblastoma, esophageal cancer, genitourinary tract cancer, malignant hypercalcemia, cervical cancer, endometrial cancer, adrenocortical cancer, and prostate cancer cancer.
“神经疾病或病症”是指神经系统和/或视觉系统的任何疾病或病症。“神经疾病或病症”包括涉及中枢神经系统(脑、脑干和小脑)、周围神经系统(包括脑神经)和自主神经系统(其部分位于中枢和周围神经系统二者中)的疾病或病症。神经病症的实例包括但不限于，头痛、木僵和昏迷、痴呆、发作、睡眠障碍、创伤、感染、新生物、神经眼科学、运动障碍、脱髓鞘病、脊髓病症、以及周围神经、肌肉和神经肌肉接点的病症。成瘾和精神病也包含在神经病症的定义中，包括但不限于，双相性精神障碍和精神分裂症。以下为可使用根据本发明的组合物和方法治疗的数种神经障碍、症状、体征和综合征的清单：获得性癫痫样失语症；急性播散性脑脊髓炎；肾上腺脑白质营养不良；年龄相关性黄斑变性；胼胝体发育不全；失认症；艾卡迪综合征；亚历山大病；阿尔珀斯病(Alpers’disease)；交叉性偏瘫；血管性痴呆；肌萎缩侧索硬化；无脑畸形；Angelma综合征；血管瘤病；缺氧；失语症；失用症；蛛网膜囊肿；蛛网膜炎；阿-基氏脑畸形(Anronl-Chiari malformation)；动静脉畸形；阿斯佩各综合征；共济失调毛细血管扩张症(ataxia telegiectasia)；注意不集中的过度反应症；孤独症；自主神经功能障碍；背痛；巴滕病；贝切特病；贝尔氏麻痹；良性自发性睑痉挛；良性灶；肌萎缩；良性颅内高血压；宾斯旺格病；睑痉挛；Bloch Sulzberger综合征；臂丛损伤；脑脓肿；脑损伤；脑肿瘤(包括多形性成胶质细胞瘤)；脊髓肿瘤；布朗-塞卡尔综合征；卡纳万病；腕管综合征；灼痛；中枢性疼痛综合征；脑桥中央髓鞘溶解；头部病症(cephalicdisorder)；脑动脉瘤；脑动脉硬化；脑萎缩；大脑性巨人症；大脑性瘫痪；夏-马-图病；化学疗法诱导的神经病和神经性疼痛；Chiari畸形；舞蹈症；慢性炎性脱髓鞘性多神经病；慢性痛；慢性区域性疼痛综合征；科-勒综合征；昏迷，包括持续性植物状态；先天性面瘫；皮质基底节变性；颅动脉炎；颅缝早闭；克雅病；累积性创伤障碍(cumulative trauma disorder)；库兴综合征(Cushing′s syndrome)；巨细胞包涵体病；巨细胞病毒感染；舞蹈眼-舞蹈脚综合征；丹-沃(DandyWalker)综合征；Dawson病；德摩西埃综合征；Dejerine-Klumke麻痹；痴呆；皮肌炎；糖尿病神经病变；弥漫性硬化；自主神经功能异常；书写困难；诵读困难；张力失常；早期幼儿癫痫性脑病；空蝶鞍综合征；脑炎；脑膨出；脑三叉神经血管瘤病；癫痫；欧勃麻痹；特发性震颤；法布里病；法尔综合征；昏厥；家族性痉挛性瘫痪；热性癫痫发作；费希尔综合征；弗里德赖希共济失调；额颞痴呆和其它“tau蛋白病变(tauopathies)”；高歇病；格斯特曼综合征；巨细胞动脉炎；巨细胞性包涵体病；球样细胞脑白质营养不良；格-巴综合征；HTLV-1相关性脊髓病；哈-斯病；颅脑损伤；头痛；偏侧面肌痉挛；遗传性痉挛性截瘫；多神经炎型遗传性共济失调；耳部带状疱疹；带状疱疹；Hirayama综合征；HIV相关性痴呆和神经病(还是AIDS的神经表现)；前脑无裂畸形；亨廷顿舞蹈病和其它聚谷氨酰胺重复疾病(polyglutamine repeat disease)；积水性无脑畸形；脑积水；皮质醇增多症；缺氧；免疫介导的脑脊髓炎；包涵体肌炎；色素失调症；婴儿植烷酸贮积病；婴儿雷夫叙姆病；婴儿性痉挛；炎性肌病；颅内囊肿；颅内高血压；Joubert综合征；基-塞(Keams-Sayre)综合征；肯尼迪病Kinsboum综合征；克-费(Klippel Feil)综合征；克拉伯病；库-韦病；库鲁病；拉福拉病；朗-爱(Lambert-Eaton)肌无力综合征；Landau-Kleffner综合征；侧髓(瓦伦伯格)综合征；学习无能；利氏病；Lennox-Gustaut综合征；累-奈综合征；脑白质营养不良；路易(Lewy)体痴呆；无脑回；闭锁综合征；Lou Gehrig病(即，运动神经元病或肌萎缩侧索硬化)；椎间盘疾病；莱姆病-神经后遗症；马-约病；巨脑(macrencephaly)；巨脑(megalencephaly)；梅-罗综合征；美尼尔症；脑膜炎；门克斯病；异染性脑白质营养不良；小头畸形；偏头痛；米勒费希尔综合征；小中风(mini-stroke)；线粒体肌病；默比厄斯综合征；单肢肌萎缩；运动神经元病；脑底异常血管网病；粘多糖症；多发性梗塞性痴呆(milti-infarct dementia)；多病灶运动神经病；多发性硬化和其它脱髓鞘病症；多系统萎缩伴随直立性低血压；p肌肉萎缩症；重症肌无力；髓鞘脱失(myelinoclastic)弥漫性硬化；婴儿肌阵挛性脑病；肌阵挛；肌病；先天性肌强直；发作性睡病；神经纤维瘤病；神经阻滞剂恶性综合征；AIDS的神经表现；狼疮的神经后遗症；神经性肌强直；神经元腊样脂褐质症；神经元迁移障碍；尼曼-皮克病；O’Sullivan-McLeod综合征；枕神经痛；隐性脊柱神经管闭合不全序列征；大田原(Ohtahara)综合征；橄榄体脑桥小脑萎缩；视性眼肌阵痉挛；视神经炎；直立性低血压；过度使用综合征；感觉异常；神经变性疾病或病症(帕金森病、亨廷顿舞蹈病、阿尔茨海默氏病、肌萎缩侧索硬化(ALS)、痴呆、多发性硬化以及其它和神经元细胞死亡有关的疾病和病症)；先天性副肌强直；类肿瘤性疾病；发作性发作(paroxysmal attacks)；帕-罗(Parry Romberg)综合征；佩-梅病；周期性瘫痪；周围神经病；疼痛性神经病和神经性疼痛；持续性植物状态；全身性发育迟缓；感光性喷嚏反射；植烷酸贮积病；皮克病；神经挟捏；垂体瘤；多肌炎；脑穿通畸形；小儿麻痹症后期综合征；疱疹后神经痛；感染后脑脊髓炎；直立性低血压；普-韦(Prader-Willi)综合征；原发性侧索硬化；朊病毒病；进行性一侧面萎缩；进行性多灶性白质脑病；进行性硬化性灰质萎缩；进行性核上性麻痹；脑假瘤；拉姆齐·亨特综合征(I型和11型)；拉斯马森(Rasmussen)脑炎；反射性交感神经营养不良综合征；雷夫叙姆病；反复性运动障碍；反复性应激损伤；腿多动综合征；反转录病毒相关性脊髓病；雷特综合征；雷耶综合征；舞蹈病；桑德霍夫病；谢耳德病；脑裂；中隔-视神经发育不良(septo-optic dysplasia)；惊吓婴儿综合征；带状疱疹；希-德综合征；斯耶格伦综合征；睡眠性呼吸暂停；索托斯综合征(Soto′s syndrome)；痉挛状态；脊柱裂；脊髓损伤；脊髓瘤；脊髓性肌萎缩；僵人综合征(Stiff-Person syndrome)；中风；斯-韦综合征；亚急性硬化性全脑炎；皮层下动脉硬化性脑病；西德纳姆舞蹈病；晕厥；脊髓空洞症；迟发性运动障碍；泰-萨克斯病；颞动脉炎；脊髓栓系综合征(tethered spinal cord syndrome)；肌强直性白内障；胸廓出口综合征；三叉神经痛；Todd麻痹；图雷特综合症；短暂性脑缺血发作；传染性海绵状脑病；横贯性脊髓炎；外伤性脑损伤；震颤；三叉神经痛；热带痉挛性轻截瘫；结节性硬化症；血管性痴呆(多发梗塞性痴呆)；血管炎，包括颞动脉炎；希-林(Von Hippel-Lindau)病；瓦伦伯格综合征；韦-霍(Werdnig-Hoffman)病；韦斯特综合征；急性颈部扭伤(whiplash)；威廉斯综合征；Wildon病；以及泽尔韦格综合征。"Neurologic disease or disorder" refers to any disease or disorder of the nervous system and/or visual system. "Neurological diseases or disorders" include diseases or disorders involving the central nervous system (brain, brainstem, and cerebellum), peripheral nervous system (including cranial nerves), and autonomic nervous system (parts of which are located in both the central and peripheral nervous systems). Examples of neurological disorders include, but are not limited to, headache, stupor and coma, dementia, seizures, sleep disturbances, trauma, infection, neoplasm, neuro-ophthalmology, movement disorders, demyelinating diseases, spinal cord disorders, and peripheral nerve, muscle and disorders of the neuromuscular junction. Addiction and psychosis are also included in the definition of neurological conditions, including, but not limited to, bipolar disorder and schizophrenia. The following is a list of several neurological disorders, symptoms, signs and syndromes that can be treated using the compositions and methods according to the present invention: acquired epileptiform aphasia; acute disseminated encephalomyelitis; adrenoleukodystrophy; age Associated macular degeneration; hypoplasia of the corpus callosum; agnosia; Ecardi syndrome; Alexander disease; Alpers' disease; crossed hemiplegia; vascular dementia; amyotrophic lateral sclerosis; anencephaly; Angelma syndrome; Angiomatosis; Hypoxia; Aphasia; Apraxia; Arachnoid cyst; Arachnoiditis; Anronl-Chiari malformation; Arteriovenous malformation; Asperger syndrome; Ataxia telegiectasia; inattentive hyperreactive disorder; autism; autonomic dysfunction; back pain; Batten's disease; Behcet's disease; Bell's palsy; benign spontaneous blepharospasm; benign Foci; Muscular atrophy; Benign intracranial hypertension; Binswanger disease; Blepharospasm; Bloch Sulzberger syndrome; Brachial plexus injury; Brain abscess; Brain injury; Brain tumors (including glioblastoma multiforme); Spinal cord Tumor; Brown-Seccal syndrome; Canavan disease; carpal tunnel syndrome; causalgia; central pain syndrome; central pontine myelination; cephalic disorder; cerebral aneurysm; cerebral arteriosclerosis; brain atrophy Cerebral gigantism; Cerebral palsy; Schia-Marie-Too disease; Chemotherapy-induced neuropathy and neuropathic pain; Chiari malformation; Chorea; Chronic inflammatory demyelinating polyneuropathy; Chronic pain; Chronic regional pain syndrome; Kohler syndrome; coma, including persistent vegetative state; congenital facial paralysis; corticobasal degeneration; cranial arteritis; craniosynostosis; Creutzfeldt-Jakob disease; cumulative trauma disorder; library Cushing's syndrome; giant cell inclusion body disease; cytomegalovirus infection; dancing eye-dancing feet syndrome; Dandy Walker syndrome; Dawson disease; Demosier syndrome; Dejerine-Klumke Paralysis; dementia; dermatomyositis; diabetic neuropathy; diffuse sclerosis; autonomic dysfunction; dysgraphia; dyslexia; dystonia; early childhood epileptic encephalopathy; empty sella syndrome; encephalitis; encephalocele; encephalopathy Trigeminal angiomatosis; epilepsy; Erber's palsy; essential tremor; Fabry disease; Fall syndrome; syncope; familial spastic paralysis; febrile seizures; Fisher syndrome; Friedrei His ataxia; frontotemporal dementia and other "tauopathies"; Gaucher disease; Gerstmann syndrome; giant cell arteritis; giant cell inclusion disease; globular cell leukodystrophy; Guerrilla-Barr syndrome; HTLV-1-related myelopathy; Haas disease; craniocerebral injury; headache ; Hemifacial spasms; Hereditary spastic paraplegia; Hereditary ataxia of the polyneuritis type; Herpes zoster auris; Herpes zoster; Hirayama syndrome; Holoprosencephaly; Huntington's disease and other polyglutamine repeat diseases; hydroanencephaly; hydrocephalus; hypercortisolism; hypoxia; immune-mediated encephalomyelitis; Inclusion body myositis; pigmentary disorders; infantile phytanic acid storage disease; infantile Refsum disease; infantile spasms; inflammatory myopathy; intracranial cyst; intracranial hypertension; Joubert syndrome; Keams-Sayre Syndrome; Kennedy Disease Kinsboum Syndrome; Klippel Feil Syndrome; Asthenia Syndrome; Landau-Kleffner Syndrome; Lateral Myeloid (Wallenberg) Syndrome; Learning Disability; Leigh's Disease; Lennox-Gustaut Syndrome; Lewy Syndrome; Leukodystrophy; Lewy Body Dementia; lissencephaly; locked-in syndrome; Lou Gehrig's disease (ie, motor neuron disease or amyotrophic lateral sclerosis); intervertebral disc disease; Cerebral (megalencephaly); May-Row syndrome; Meniere's disease; meningitis; Menkes' disease; metachromatic leukodystrophy; microcephaly; migraine; Miller-Fischer syndrome; mini-stroke); mitochondrial myopathy; Möbius syndrome; muscular atrophy of one limb; motor neuron disease; abnormal vascular network disease at the base of the brain; mucopolysaccharidosis; Focal motor neuropathy; multiple sclerosis and other demyelinating disorders; multiple system atrophy with orthostatic hypotension; muscular dystrophy; myasthenia gravis; myelinoclastic diffuse sclerosis; infantile myoclonic encephalopathy; Myoclonus; myopathy; myotonia congenita; narcolepsy; neurofibromatosis; neuroleptic malignant syndrome; neurological manifestations of AIDS; neurological sequelae of lupus; neuromyotonia; neuronal ceroid Brown matter; neuronal migration disorder; Niemann-Pick disease; O'Sullivan-McLeod syndrome; occipital neuralgia; recessive spinal neural tube incompetence sequence; Ohtahara syndrome; olivopontocerebellar atrophy ; optic ophthalmospasm; optic neuritis; orthostatic hypotension; overuse syndrome; paresthesias; neurodegenerative diseases or conditions (Parkinson's disease, Huntington's disease, Alzheimer's disease, ALS (ALS), dementia, multiple sclerosis, and other diseases and diseases associated with neuronal cell death paramyotonia congenita; neoplastic disorders; paroxysmal attacks; Parry Romberg syndrome; Pey-May disease; periodic paralysis; peripheral neuropathy; painful neuropathy and neuropathic neuropathy Pain; persistent vegetative state; generalized developmental delay; photosensitive sneeze reflex; phytanic acid storage disease; Pick's disease; nerve pinch; pituitary tumor; polymyositis; Postherpetic neuralgia; postinfectious encephalomyelitis; orthostatic hypotension; Prader-Willi syndrome; primary lateral sclerosis; prion diseases; progressive lateral atrophy; progressive multifocal leukoencephalopathy ; progressive sclerosing gray matter atrophy; progressive supranuclear palsy; cerebral pseudotumor; Ramsay Hunter syndrome (types 1 and 11); Rasmussen encephalitis; reflex sympathetic neurotrophy Adverse syndrome; Refsum disease; repetitive movement disorder; repetitive stress injury; restless leg syndrome; retrovirus-associated myelopathy; Rett syndrome; Reye syndrome; chorea; mulberry Derhoffer disease; Shelder disease; split brain; septo-optic dysplasia; startled baby syndrome; herpes zoster; Schneider syndrome; Sjogren syndrome; Apnea; Soto's syndrome; Spasticity; Spina bifida; Spinal cord injury; Spinal myeloma; Spinal muscular atrophy; Stiff-Person syndrome; Stroke; ; subacute sclerosing panencephalitis; subcortical arteriosclerotic encephalopathy; Sydenham chorea; syncope; syringomyelia; tardive dyskinesia; Tay-Sachs disease; temporal arteritis; tethered cord syndrome ( tethered spinal cord syndrome); myotonic cataract; thoracic outlet syndrome; trigeminal neuralgia; Todd's palsy; Tourette's syndrome; transient ischemic attack; transmissible spongiform encephalopathy; transverse myelitis; traumatic brain injury Injury; tremor; trigeminal neuralgia; tropical spastic paraplegia; tuberous sclerosis; vascular dementia (multi-infarct dementia); vasculitis, including temporal arteritis; Von Hippel-Lindau disease; Lemberg Syndrome; Werdnig-Hoffman Disease; West Syndrome; Acute Whiplash; Williams Syndrome; Wildon Disease; and Zellweger Syndrome.
“炎症”是指全身性炎性病况以及局部地与单核细胞、白细胞和/或中性粒细胞的迁移和吸引有关的病况。炎症的实例包括但不限于，因以下引发的炎症：感染致病生物(包括革兰氏阳性菌、革兰氏阴性菌、病毒、真菌、以及寄生物，例如原生动物和蠕虫)、移植排斥(包括实质器官的排斥，例如肾、肝、心、肺或角膜，以及骨髓移植的排斥，包括移植物抗宿主病(GVHD))或者局限性慢性或急性自身免疫反应或变态反应。自身免疫性疾病包括急性肾小球肾炎；类风湿性或反应性关节炎；慢性肾小球肾炎；炎性肠病，例如克罗恩氏病、溃疡性结肠炎和坏死性小肠结肠炎；与粒细胞输注有关的综合征；炎性皮肤病，例如接触性皮炎、特应性皮炎、银屑病；系统性红斑狼疮(SLE)；自身免疫性甲状腺炎、多发性硬化、以及糖尿病的某些形式、或任何其它自体免疫状态(其中受试者自身免疫系统的攻击导致病理性组织破坏)。变态反应包括变应性哮喘、慢性支气管炎、急性和迟发型超敏反应。全身性炎性疾病状态包括与创伤、烧伤、缺血事件后的再灌注(例如在心、脑、肠或外周脉管系统中的血栓形成事件，包括心肌梗死和中风)、脓毒症、ARDS或多器官功能障碍综合征相关的炎症。炎性细胞募集也在粥样硬化斑块中发生。炎症包括但不限于，非何杰金淋巴瘤、韦格纳肉芽肿病、桥本甲状腺炎、肝细胞癌、胸腺萎缩、慢性胰腺炎、类风湿性关节炎、反应性淋巴样增生、骨关节炎、溃疡性结肠炎、乳头状癌、克罗恩氏病、溃疡性结肠炎、急性胆囊炎、慢性胆囊炎、肝硬化、慢性涎腺炎、腹膜炎、急性胰腺炎、慢性胰腺炎、慢性胃炎、子宫肌腺病、子宫内膜异位、急性子宫颈炎、慢性子宫颈炎、淋巴样增生、多发性硬化、继发于特发性血小板减少性紫癜的肥大、原发性IgA肾病、系统性红斑狼疮、银屑病、肺气肿、慢性肾盂肾炎、以及慢性膀胱炎。"Inflammation" refers to systemic inflammatory conditions as well as conditions locally associated with the migration and attraction of monocytes, leukocytes and/or neutrophils. Examples of inflammation include, but are not limited to, inflammation resulting from infection with pathogenic organisms (including Gram-positive bacteria, Gram-negative bacteria, viruses, fungi, and parasites such as protozoa and helminths), transplant rejection ( Includes rejection of solid organs, such as kidney, liver, heart, lung, or cornea, and rejection of bone marrow transplants, including graft-versus-host disease (GVHD)) or localized chronic or acute autoimmune or allergic reactions. Autoimmune diseases include acute glomerulonephritis; rheumatoid or reactive arthritis; chronic glomerulonephritis; inflammatory bowel diseases such as Crohn's disease, ulcerative colitis, and necrotizing enterocolitis; Syndromes associated with granulocyte infusion; inflammatory skin diseases such as contact dermatitis, atopic dermatitis, psoriasis; systemic lupus erythematosus (SLE); autoimmune thyroiditis, multiple sclerosis, and some forms of diabetes These forms, or any other autoimmune state in which attack by the subject's own immune system results in pathological tissue destruction. Allergic reactions include allergic asthma, chronic bronchitis, acute and delayed hypersensitivity reactions. Systemic inflammatory disease states include those associated with trauma, burns, reperfusion following ischemic events (eg, thrombotic events in the heart, brain, intestine, or peripheral vasculature, including myocardial infarction and stroke), sepsis, ARDS, or Inflammation associated with multiple organ dysfunction syndrome. Inflammatory cell recruitment also occurs in atherosclerotic plaques. Inflammation includes, but is not limited to, non-Hodgkin's lymphoma, Wegener's granulomatosis, Hashimoto's thyroiditis, hepatocellular carcinoma, thymic atrophy, chronic pancreatitis, rheumatoid arthritis, reactive lymphoid hyperplasia, osteoarticular Inflammation, ulcerative colitis, papillary carcinoma, Crohn's disease, ulcerative colitis, acute cholecystitis, chronic cholecystitis, liver cirrhosis, chronic sialadenitis, peritonitis, acute pancreatitis, chronic pancreatitis, chronic gastritis , adenomyosis, endometriosis, acute cervicitis, chronic cervicitis, lymphoid hyperplasia, multiple sclerosis, hypertrophy secondary to idiopathic thrombocytopenic purpura, primary IgA nephropathy, systemic erythema Lupus, psoriasis, emphysema, chronic pyelonephritis, and chronic cystitis.
心血管疾病或病症包括可引起缺血或者由心脏的再灌注引发的那些病症。实例包括但不限于，动脉粥样硬化、冠状动脉疾病、肉芽肿性心肌炎、慢性心肌炎(非肉芽肿性)、原发性肥大性心肌病、外周动脉病(PAD)、中风、心绞痛、心肌梗死、由心搏停止引发的心血管组织损伤、由心脏旁流引发的心血管组织损伤、心源性休克，以及相关的病况，所述病况会是本领域普通技术人员已知的，或涉及心脏或脉管系统的机能障碍或组织损伤，特别是但不限于，与抗病毒基因激活有关的组织损伤。CVS疾病包括但不限于，动脉粥样硬化、肉芽肿性心肌炎、心肌梗死、继发于瓣膜性心脏病的心肌纤维化、无梗死形成的心肌纤维化、原发性肥大性心肌病、以及慢性心肌炎(非肉芽肿性)。Cardiovascular diseases or disorders include those that can cause ischemia or are triggered by reperfusion of the heart. Examples include, but are not limited to, atherosclerosis, coronary artery disease, granulomatous myocarditis, chronic myocarditis (non-granulomatous), primary hypertrophic cardiomyopathy, peripheral arterial disease (PAD), stroke, angina, myocardial infarction , cardiovascular tissue damage from asystole, cardiovascular tissue damage from bypass of the heart, cardiogenic shock, and related conditions that would be known to those of ordinary skill in the art, or involve the heart or dysfunction of the vasculature or tissue damage, particularly, but not limited to, tissue damage associated with activation of antiviral genes. CVS diseases include, but are not limited to, atherosclerosis, granulomatous myocarditis, myocardial infarction, myocardial fibrosis secondary to valvular heart disease, myocardial fibrosis without infarction, primary hypertrophic cardiomyopathy, and chronic Myocarditis (non-granulomatous).
造成患者疾病的病毒(一种或复数种)的实例包括：疱疹病毒，例如，α-疱疹病毒(例如单纯疱疹病毒1(HSV-1)；单纯疱疹病毒2(HSV-2)；水痘带状疱疹病毒(VZV))、β-疱疹病毒(例如巨细胞病毒(CMV)；疱疹病毒6(HHV-6))、γ-疱疹病毒(例如非洲淋巴细胞瘤病毒(EBV)；疱疹病毒8(HHV-8))、肝炎病毒(例如甲型肝炎病毒；乙型肝炎病毒；丙型肝炎病毒；丁型肝炎病毒；戊型肝炎病毒)、逆转录病毒(例如人免疫缺陷病毒1(HIV-1))、正粘病毒(例如甲、乙和丙型流感病毒)、副粘病毒、呼吸道合胞病毒(RSV)、副流感病毒(PI)(例如腮腺炎病毒、麻疹病毒)、披膜病毒(例如风疹病毒)、小核糖核酸病毒(例如肠道病毒、鼻病毒；冠状病毒)、乳多空病毒(例如人乳头瘤病毒(HPV)；多瘤病毒(BKV和ICV)；胃肠炎病毒)、纤丝病毒科、布尼安病毒科、弹状病毒科、黄病毒科。Examples of virus(s) causing disease in a patient include: herpes viruses, e.g., alpha-herpes viruses (e.g. herpes simplex virus 1 (HSV-1); herpes simplex virus 2 (HSV-2); varicella zoster Herpesviruses (VZV)), beta-herpesviruses (e.g., cytomegalovirus (CMV); herpesvirus 6 (HHV-6)), gamma-herpesviruses (e.g., African lymphoma virus (EBV); -8)), hepatitis viruses (such as hepatitis A virus; hepatitis B virus; hepatitis C virus; hepatitis D virus; hepatitis E virus), retroviruses (such as human immunodeficiency virus 1 (HIV-1) ), orthomyxoviruses (e.g. influenza A, B, and C), paramyxoviruses, respiratory syncytial virus (RSV), parainfluenza viruses (PI) (e.g. mumps, measles), togaviruses (e.g. rubella virus), picornaviruses (e.g., enteroviruses, rhinoviruses; coronaviruses), papovaviruses (e.g., human papillomaviruses (HPV); polyomaviruses (BKV and ICV); gastroenteritis viruses), Filoviridae, Bunyanviridae, Rhabdoviridae, Flaviviridae.
造成细菌疾病的细菌的实例包括：金黄色葡萄球菌：菌株包括甲氧西林抗性的(MRSA)，甲氧西林-万古霉素抗性的(VMRSA)和万古霉素中间体抗性的(VISA)；表皮葡萄球菌；粪肠球菌和屎肠球菌：菌株包括万古霉素抗性的(VRE)；肺炎链球菌；铜绿假单孢菌；洋葱伯霍尔德杆菌；嗜麦芽黄单胞菌；大肠杆菌；肠杆菌属；肺炎克雷伯菌；沙门菌属。Examples of bacteria that cause bacterial disease include: Staphylococcus aureus: Strains include methicillin-resistant (MRSA), methicillin-vancomycin resistant (VMRSA) and vancomycin intermediate resistant (VISA ); Staphylococcus epidermidis; Enterococcus faecalis and Enterococcus faecium: strains including vancomycin-resistant (VRE); Streptococcus pneumoniae; Pseudomonas aeruginosa; Burholderia cepacia; Xanthomonas maltophilia; Escherichia coli; Enterobacter; Klebsiella pneumoniae; Salmonella.
多核苷酸和寡核苷酸组合物和分子Polynucleotide and oligonucleotide compositions and molecules
靶物target
在一个实施方案中，所述靶物包含抗病毒基因的核酸序列，包括但不限于：与抗病毒基因有关的有义和/或反义非编码和/或编码序列。In one embodiment, the target comprises nucleic acid sequences of antiviral genes, including but not limited to: sense and/or antisense non-coding and/or coding sequences related to antiviral genes.
在一个实施方案中，所述靶物包含RIG1的核酸序列，包括但不限于：与RIG1基因有关的有义和/或反义非编码和/或编码序列。In one embodiment, the target comprises the nucleic acid sequence of RIG1, including but not limited to: sense and/or antisense non-coding and/or coding sequences related to the RIG1 gene.
在一个实施方案中，所述靶物包含MDA5的核酸序列，包括但不限于：与MDA5基因有关的有义和/或反义非编码和/或编码序列In one embodiment, the target comprises the nucleic acid sequence of MDA5, including but not limited to: sense and/or antisense non-coding and/or coding sequences related to the MDA5 gene
在一个实施方案中，所述靶物包含IFNA1的核酸序列，包括但不限于：与IFNA1基因有关的有义和/或反义非编码和/或编码序列In one embodiment, the target comprises the nucleic acid sequence of IFNA1, including but not limited to: sense and/or antisense non-coding and/or coding sequences related to the IFNA1 gene
视黄酸可诱导的基因I产物(RIG-I)已经被鉴别为RNA病毒感染的细胞传感器，所述RNA病毒感染会导致β干扰素(IFN-B)诱导。但是，已知许多病毒会编码抑制IFN-B生产的病毒产物。在甲型流感病毒的情况下，病毒的非结构蛋白1(NS1)会如下阻止IFN-B启动子的诱导：通过抑制包括IRF-3在内的转录因子的活化，所述转录因子参与IFN-B转录活化。NS1的抑制性质似乎至少部分地归因于它与双链RNA(dsRNA)的结合，所述结合导致RIG-I活化的该病毒介体的隔离。The retinoic acid inducible gene I product (RIG-I) has been identified as a cellular sensor of RNA virus infection that leads to induction of interferon beta (IFN-B). However, many viruses are known to encode viral products that inhibit IFN-B production. In the case of influenza A virus, the viral nonstructural protein 1 (NS1) prevents the induction of the IFN-B promoter by inhibiting the activation of transcription factors, including IRF-3, involved in IFN- B Transcription activation. The inhibitory properties of NS1 appear to be due at least in part to its binding to double-stranded RNA (dsRNA), which leads to the sequestration of this viral mediator of RIG-I activation.
视黄酸可诱导的基因I(RIG-I)和黑素瘤分化相关基因5(MDA5)是细胞质DEx(D/H)盒解螺旋酶，所述酶可以检测细胞内的病毒产物，诸如基因组RNA(vRNA)，以发出在受感染的细胞中生产IFN-/～的信号。每次发出信号是通过同型的天冬氨酸特异性半胱氨酸蛋白酶活化和募集结构域(CARD)与干扰素启动子-刺激因子1(IPS-I)接头蛋白的相互作用来实现，其将RIG-I和MDA5募集到线粒体的外膜，作为大分子信号传递复合体的一部分，所述复合体用于激活下游干扰素调节因子(IRF)和诱导IFN-/～和ISG表达的其它转录因子。尽管RIG-I和MDA5可能具有类似的信号传递特征和结构同源性，日益增多的证据表明，这2种解螺旋酶可能辨别不同的配体，以触发对RNA病毒的先天免疫应答。RIG-I的信号传递在感染期间被许多RNA病毒触发，和被在体外转录的合成RNA的存在触发。最近，RIG-I已经涉入包含5′三磷酸酯末端的RNA部分的识别，或呈现复杂的二级结构的RNA的识别。相比而言，MDA5的信号传递独特地在微小核糖核酸病毒感染过程中被触发，或在由肌苷和胞嘧啶的对合链组成的合成RNA聚合物聚(I:C)存在下被触发。Retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5) are cytoplasmic DEx(D/H) box helicases that detect intracellular viral products such as genomic RNA (vRNA) to signal the production of IFN-/~ in infected cells. Each signaling occurs through the interaction of the homotype caspase activation and recruitment domain (CARD) with the interferon promoter-stimulatory factor 1 (IPS-I) adapter protein, which Recruits RIG-I and MDA5 to the outer membrane of mitochondria as part of a macromolecular signaling complex for activation of downstream interferon regulatory factors (IRFs) and other transcriptional induction of IFN-/~ and ISG expression factor. Although RIG-I and MDA5 may share similar signaling profiles and structural homology, accumulating evidence suggests that these 2 helicases may recognize distinct ligands to trigger innate immune responses to RNA viruses. RIG-I signaling is triggered by many RNA viruses during infection, and by the presence of synthetic RNA transcribed in vitro. More recently, RIG-I has been implicated in the recognition of portions of RNA containing 5' triphosphate termini, or of RNAs exhibiting complex secondary structures. In contrast, signaling of MDA5 is uniquely triggered during picornavirus infection or in the presence of poly(I:C), a synthetic RNA polymer composed of inosine and cytosine fused strands .
通过适当的药理学操纵，可以将人黑素瘤细胞重新程序化成最终分化，且不可逆地丧失增殖能力。减法杂交将黑素瘤分化相关基因-5(mda-5)鉴别为在分化、癌症逆转和程序化细胞死亡(细胞凋亡)过程中诱导的基因。该基因含有天冬氨酸特异性半胱氨酸蛋白酶募集结构域和假定的DexH组RNA解螺旋酶结构域。MDA-5的非典型解螺旋酶基序偏离共有序列，但是在克隆的和假定的蛋白的潜在新组中是非常保守的。mda-5是可由IFN和肿瘤坏死因子-a诱导的早期应答基因，主要对IFN-～做出应答。瑞香素对蛋白激酶C的活化会进一步增加由IFN-～诱导的mda-5表达。mda-5的表达由IFN-～转录地控制，MDA-5蛋白定位于细胞质中。mda-5显示出RNA-依赖性的ATP酶活性，且mda5在人黑素瘤细胞中的异位表达会抑制集落形成。在这些背景下，mda-5可能起IFN-诱导的生长抑制和/或细胞凋亡的介体的功能。MDA-5是一种双链RNA-依赖性的ATP酶，其含有天冬氨酸特异性半胱氨酸蛋白酶募集结构域和RNA解螺旋酶基序，它们经证实与在人黑素瘤细胞中的生长和分化有关。Through appropriate pharmacological manipulation, human melanoma cells can be reprogrammed to terminal differentiation with irreversible loss of proliferative capacity. Subtractive hybridization identified melanoma differentiation-associated gene-5 (mda-5) as a gene induced during differentiation, cancer reversal and programmed cell death (apoptosis). This gene contains a caspase-specific protease recruitment domain and a putative DexH group RNA helicase domain. The atypical helicase motif of MDA-5 deviates from the consensus sequence, but is well conserved in potentially new groups of cloned and putative proteins. mda-5 is an early response gene induced by IFN and tumor necrosis factor-a, and it mainly responds to IFN-~. The activation of protein kinase C by daphnetin will further increase the expression of mda-5 induced by IFN-~. The expression of mda-5 is transcriptionally controlled by IFN-~, and the MDA-5 protein is localized in the cytoplasm. mda-5 exhibits RNA-dependent ATPase activity, and ectopic expression of mda5 in human melanoma cells inhibits colony formation. In these contexts, mda-5 may function as a mediator of IFN-induced growth inhibition and/or apoptosis. MDA-5 is a double-stranded RNA-dependent ATPase containing a caspase-specific protease-recruiting domain and an RNA helicase motif that was demonstrated to be associated with human melanoma cells related to growth and differentiation.
关于干扰素(IFN)的抗病毒作用的分子基础以及病毒对抗IFN的作用所涉及的策略的理解，已经取得了进展。此外，在IFN系统已经显著促进我们对病毒学和分子细胞生物学的多个领域(其范围从信号转导途径至转录和翻译控制的生化机制，至病毒致病机制的分子基础)的理解的同时，取得了进展。IFN是经批准的治疗剂，且已经从基础研究实验室进入临床。在IFN的抗病毒作用中重要的IFN-诱导的蛋白是：RNA-依赖性的蛋白激酶(PKR)、2′，5′-寡腺苷酸合成酶(OAS)和RNA酶L和Mx蛋白GTP酶。双链RNA在调节蛋白磷酸化和RNA降解(分别由IFN-可诱导的PKR激酶和2′-5′-寡腺苷酸-依赖性的RNA酶L催化)中起中枢作用，且也在IFN-可诱导的RNA-特异性的腺苷脱氨酶(ADAR1)的RNA编辑中起中枢作用。IFN也会诱导可诱导的一氧化氮合酶(iNOS2)和主要组织相容性复合物I类和II类蛋白的形成，它们都在对感染的免疫应答中起重要作用。通过微阵列分析，已经鉴别出几个额外的基因，所述基因的表达特性响应于IFN治疗和病毒感染而改变。cDNA和基因组克隆对于IFN系统的许多组分(包括IFN-α、IFN-β和IFN-γ、它们的受体、Jak和Stat和IRF信号转导组分和蛋白诸如PKR、2′、5′-OAS、Mx和ADAR，它们的表达受IFN调节)的可用性，已经允许制备突变型蛋白、过表达所述蛋白的不同形式的细胞以及这样的动物：它们在所述动物中的表达已经通过靶向基因破坏而破坏。这些IFN系统试剂在细胞培养物中和在完整动物中的应用，继续为我们对病毒-宿主相互作用和细胞抗病毒应答的理解提供重要贡献。Progress has been made in the understanding of the molecular basis of the antiviral effects of interferons (IFNs) and the strategies involved in the actions of viruses against IFNs. Furthermore, the IFN system has significantly advanced our understanding of multiple areas of virology and molecular cell biology, which range from signal transduction pathways, to biochemical mechanisms of transcriptional and translational control, to the molecular basis of viral pathogenic mechanisms. Meanwhile, progress has been made. IFN is an approved therapeutic agent that has moved from the basic research laboratory to the clinic. The IFN-induced proteins important in the antiviral action of IFN are: RNA-dependent protein kinase (PKR), 2',5'-oligoadenylate synthase (OAS) and RNase L and the Mx protein GTP enzyme. Double-stranded RNA plays a central role in regulating protein phosphorylation and RNA degradation (catalyzed by IFN-inducible PKR kinase and 2′-5′-oligoadenylate-dependent RNase L, respectively), and is also - Inducible RNA-specific adenosine deaminase (ADAR1) plays a central role in RNA editing. IFN also induces the formation of inducible nitric oxide synthase (iNOS2) and major histocompatibility complex class I and class II proteins, both of which play important roles in the immune response to infection. Through microarray analysis, several additional genes have been identified whose expression profiles are altered in response to IFN treatment and viral infection. cDNA and genomic cloning for many components of the IFN system (including IFN-α, IFN-β and IFN-γ, their receptors, Jak and Stat and IRF signaling components and proteins such as PKR, 2′, 5′ -Availability of OAS, Mx and ADAR, whose expression is regulated by IFN), has allowed the production of mutant proteins, cells overexpressing different forms of said proteins, and animals in which their expression has been regulated by target Destruction towards genetic destruction. The application of these IFN system reagents in cell culture and in intact animals continues to provide important contributions to our understanding of virus-host interactions and cellular antiviral responses.
干扰素(IFN)在病毒感染过程中参与许多免疫相互作用，并促成先天性的和适应性的抗病毒机理的诱导和调节。IFN在病毒感染的结果中起关键作用，如受体IFNAR-2和IFNGR缺陷型小鼠对不同病毒的抗性受损所证实的。在病毒感染过程中，IFN作为先天性和适应性抗病毒机理的诱导物、调节物和效应物参与许多免疫相互作用。当病毒因子(诸如外壳糖蛋白、CpG DNA或dsRNA)与细胞的模式-识别受体(PRR)(诸如甘露糖受体、toll-样受体(TLR)和细胞溶质受器)相互作用时，会快速地生成IFN-α/β。这些宿主-病毒相互作用向下游发出信号，以活化实现IFN-α/β基因表达所需的转录因子。它们包括IFN调节因子-3(IRF-3)、IRF-5、IRF-7、c-Jun/ATF-2和NF-κB。相比而言，IFN-γ由受体-介导的刺激来诱导，或响应于早期生产的细胞因子(包括白介素-2(IL-12)、IL-18和IFN-α/β)而诱导，或被通过T细胞受体(TCR)或天然杀伤(NK)细胞受体的刺激诱导。IFN通过跨膜受体发出信号，主要活化Jak-Stat途径，但是也活化其它信号转导途径。细胞因子和TCR-诱导的IFN-γ表达使用独特的信号转导途径，其中涉及诸如NFAT、Stats和NF-κB等转录因子。这导致许多原有的抗病毒因子的诱导和活化，所述抗病毒因子例如RNA-活化的蛋白激酶(PKR)、2-5A系统、Mx蛋白和几个细胞凋亡途径。另外，IFN会调节先天性和适应性免疫的不同方面。因而，IFNα/β和IFN-γ会通过增加抗原呈递、细胞运输以及细胞分化和表达特性，影响巨噬细胞、NK细胞、树突细胞(DC)和T细胞的活性，最终导致增强的抗病毒效应物功能。Interferon (IFN) participates in many immune interactions during viral infection and contributes to the induction and regulation of innate and adaptive antiviral mechanisms. IFN plays a key role in the outcome of viral infection, as evidenced by the impaired resistance of mice deficient for the receptors IFNAR-2 and IFNGR to different viruses. During viral infection, IFNs are involved in many immune interactions as inducers, regulators and effectors of innate and adaptive antiviral mechanisms. When viral factors, such as coat glycoproteins, CpG DNA or dsRNA, interact with the cell's pattern-recognition receptors (PRRs), such as mannose receptors, toll-like receptors (TLRs), and cytosolic receptors, Rapid production of IFN-α/β. These host-virus interactions signal downstream to activate transcription factors required to achieve IFN-α/β gene expression. They include IFN regulatory factor-3 (IRF-3), IRF-5, IRF-7, c-Jun/ATF-2 and NF-κΒ. In contrast, IFN-γ is induced by receptor-mediated stimulation, or in response to early production of cytokines including interleukin-2 (IL-12), IL-18, and IFN-α/β , or induced by stimulation through T cell receptor (TCR) or natural killer (NK) cell receptors. IFN signals through transmembrane receptors, primarily activating the Jak-Stat pathway, but also activating other signal transduction pathways. Cytokine and TCR-induced IFN-γ expression uses a unique signal transduction pathway involving transcription factors such as NFAT, Stats and NF-κB. This results in the induction and activation of many native antiviral factors such as RNA-activated protein kinase (PKR), the 2-5A system, Mx proteins and several apoptotic pathways. In addition, IFNs regulate different aspects of innate and adaptive immunity. Thus, IFNα/β and IFN-γ affect the activity of macrophages, NK cells, dendritic cells (DCs) and T cells by increasing antigen presentation, cell trafficking, and cell differentiation and expression properties, ultimately leading to enhanced antiviral Effector function.
在有些实施方案中，使用反义寡核苷酸来预防或治疗与抗病毒基因家族成员有关的疾病或障碍。使用细胞/组织(其从使用反义化合物得到的干细胞再生)可以治疗的示例性的抗病毒基因介导的疾病和障碍包括：癌症，炎性疾病，由传染剂(例如，包括，病毒、细菌、真菌、原生动物等)造成的疾病、障碍或病症，病毒病，炎症，关节炎，银屑病，神经学疾病或障碍，免疫系统疾病或障碍，自身免疫疾病或障碍，免疫缺陷病，变态反应，银屑病，神经学疾病，肾疾病或障碍，心血管疾病或障碍，肌肉疾病或障碍，动脉粥样硬化，糖尿病，肝脏疾病或障碍和传染病。In some embodiments, antisense oligonucleotides are used to prevent or treat diseases or disorders associated with members of antiviral gene families. Exemplary antiviral gene-mediated diseases and disorders that can be treated using cells/tissues regenerated from stem cells using antisense compounds include: cancer, inflammatory diseases, , fungi, protozoa, etc.), viral disease, inflammation, arthritis, psoriasis, neurological disease or disorder, immune system disease or disorder, autoimmune disease or disorder, immunodeficiency disease, metamorphosis reactions, psoriasis, neurological disease, renal disease or disorder, cardiovascular disease or disorder, muscle disease or disorder, atherosclerosis, diabetes, liver disease or disorder, and infectious disease.
在另一个实施方案中，所述反义寡核苷酸会在患者中调节抗病毒基因的表达、体内量和/或功能，所述患者遭受与抗病毒基因有关的疾病或障碍或处于发展所述疾病或障碍的危险中。In another embodiment, the antisense oligonucleotides will modulate the expression, amount and/or function of the antiviral gene in a patient suffering from a disease or disorder associated with the antiviral gene or at a developmental stage. at risk for the disease or disorder described above.
在另一个实施例中，人变性肺病毒(HMPV)是一种最近发现的病原体，其在幼小婴儿、老年人和免疫受损的患者中造成大比例的呼吸道感染。RIG-I表达和/或功能的调控，会通过不同的机制调节病毒感染，所述机制包括例如，病毒基因组序列破坏。RIG-I反义寡核苷酸因而在抗病毒剂的开发中具有极大的治疗价值。In another example, human metapneumovirus (HMPV) is a recently discovered pathogen that causes a large proportion of respiratory tract infections in young infants, the elderly, and immunocompromised patients. Modulation of RIG-I expression and/or function regulates viral infection through different mechanisms including, for example, disruption of the viral genome sequence. RIG-I antisense oligonucleotides are thus of great therapeutic value in the development of antiviral agents.
在一个实施方案中，视黄酸可诱导的基因I(RIG-I)的调节会检测和破坏病毒基因组。在另一个实施方案中，视黄酸可诱导的基因I(RIG-I)的调节会预防或治疗患者的病毒感染。In one embodiment, modulation of retinoic acid inducible gene I (RIG-I) detects and destroys the viral genome. In another embodiment, modulation of retinoic acid inducible gene I (RIG-I) prevents or treats a viral infection in a patient.
在一个实施方案中，寡核苷酸对于抗病毒基因的多核苷酸(其包括但不限于非编码区)而言为特异性的。抗病毒基因靶标包括抗病毒基因的变体；抗病毒基因的突变体，包括SNP；抗病毒基因的非编码序列；等位基因、片段等。优选地，所述寡核苷酸为反义RNA分子。In one embodiment, the oligonucleotides are specific for polynucleotides of antiviral genes, including but not limited to non-coding regions. Antiviral gene targets include variants of antiviral genes; mutants of antiviral genes, including SNPs; non-coding sequences of antiviral genes; alleles, fragments, etc. Preferably, said oligonucleotide is an antisense RNA molecule.
根据本发明的实施方案，靶核酸分子不限于单独的抗病毒基因多核苷酸，而是扩展到抗病毒基因的任何同种型、受体、同源物、非编码区等。According to an embodiment of the present invention, the target nucleic acid molecule is not limited to a single antiviral gene polynucleotide, but extends to any isoform, receptor, homologue, non-coding region, etc. of an antiviral gene.
在另一个实施方案中，寡核苷酸靶向抗病毒基因靶标的天然反义序列(针对编码和非编码区的天然反义物)，所述抗病毒基因靶标包括但不限于其变体、等位基因、同源物、突变体、衍生物、片段和互补序列。优选所述寡核苷酸为反义RNA或DNA分子。In another embodiment, the oligonucleotides target natural antisense sequences (natural antisenses to coding and non-coding regions) of antiviral gene targets, including but not limited to variants thereof, Alleles, homologues, mutants, derivatives, fragments and complements. Preferably said oligonucleotide is an antisense RNA or DNA molecule.
在另一个实施方案中，本发明的寡聚化合物也包括变体，其中在所述化合物的一个或更多个核苷酸位置上存在不同的碱基。例如，如果第一个核苷酸为腺嘌呤，则可产生在此位置含有胸苷、鸟苷、胞苷或其它天然或非天然核苷酸的变体。这可在所述反义化合物的任何位置上完成。In another embodiment, the oligomeric compounds of the invention also include variants wherein a different base is present at one or more nucleotide positions of the compound. For example, if the first nucleotide is adenine, variants can be produced that contain thymidine, guanosine, cytidine, or other natural or unnatural nucleotides at this position. This can be done at any position on the antisense compound.
在有些实施方案中，反义化合物与靶标之间的同源性、序列同一性或互补性是约50％至约60％。在有些实施方案中，同源性、序列同一性或互补性是约60％至约70％。在有些实施方案中，同源性、序列同一性或互补性是约70％至约80％。在有些实施方案中，同源性、序列同一性或互补性是约80％至约90％。在有些实施方案中，同源性、序列同一性或互补性是约90％、约92％、约94％、约95％、约96％、约97％、约98％、约99％或约100％。In some embodiments, the homology, sequence identity or complementarity between the antisense compound and the target is about 50% to about 60%. In some embodiments, the homology, sequence identity or complementarity is about 60% to about 70%. In some embodiments, the homology, sequence identity or complementarity is about 70% to about 80%. In some embodiments, the homology, sequence identity or complementarity is about 80% to about 90%. In some embodiments, the homology, sequence identity or complementarity is about 90%, about 92%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%.
反义化合物在以下情况时为可特异性杂交的：所述化合物与靶核酸的结合干扰靶核酸的正常功能而引起活性损失，并且在需要特异性结合的条件下存在足够程度的互补性以避免所述反义化合物与非靶核酸序列的非特异性结合。这类条件包括，即，在体内测定或治疗性处理情况中的生理条件，以及其中在体外测定情况下进行测定的条件。An antisense compound is specifically hybridizable when the binding of the compound to the target nucleic acid interferes with the normal function of the target nucleic acid causing loss of activity, and a sufficient degree of complementarity exists under conditions requiring specific binding to avoid Non-specific binding of the antisense compound to a non-target nucleic acid sequence. Such conditions include, ie, physiological conditions in the case of in vivo assays or therapeutic treatments, as well as conditions in which the assay is performed in the case of in vitro assays.
反义化合物，不论DNA、RNA、嵌合的、取代的等等，在以下情况时为可特异性杂交的：所述化合物与靶DNA或RNA分子的结合干扰靶DNA或RNA的正常功能而引起效用损失，并且在需要特异性结合的条件下存在足够程度的互补性以避免所述反义化合物与非靶序列的非特异性结合，所述条件即在体内测定或治疗性处理情况中的生理条件下，以及在体外测定情况下在其中进行测定的条件下。Antisense compounds, whether DNA, RNA, chimeric, substituted, etc., are specifically hybridizable when the binding of the compound to the target DNA or RNA molecule interferes with the normal function of the target DNA or RNA causing loss of utility, and the presence of a sufficient degree of complementarity to avoid non-specific binding of the antisense compound to non-target sequences under conditions requiring specific binding, i.e., physiological conditions in the case of in vivo assays or therapeutic treatment , and in the case of an in vitro assay, the conditions in which the assay is performed.
在另一个实施方案中，靶向抗病毒基因会调节抗病毒基因的表达或功能，所述抗病毒基因包括但不限于：使用例如PCR、杂交等鉴定和扩增的反义序列，一个或更多个如SEQID NO：4-9所述的序列，等等。在一个实施方案中，表达或功能与对照相比被增量调节。在另一个实施方案中，表达或功能与对照相比被减量调节。In another embodiment, targeting antiviral genes modulates the expression or function of antiviral genes including, but not limited to: antisense sequences identified and amplified using, for example, PCR, hybridization, etc., one or more A plurality of sequences as set forth in SEQ ID NO: 4-9, etc. In one embodiment, expression or function is upregulated compared to a control. In another embodiment, expression or function is down-regulated compared to a control.
在另一个实施方案中，寡核苷酸包括如SEQ ID NO：10-30所述的核酸序列，包括使用例如PCR、杂交等鉴定和扩增的反义序列。这些寡核苷酸可包含一个或更多个经修饰的核苷酸、较短或较长的片段、经修饰的键等。经修饰的键或核苷酸间键的实例包括硫代磷酸酯、二硫代磷酸酯等。在另一个实施方案中，所述核苷酸包括磷衍生物。可连接到本发明的修饰的寡核苷酸中的糖或糖类似物部分的磷衍生物(或经修饰的磷酸基)，可为单磷酸酯、二磷酸酯、三磷酸酯、烷基磷酸酯、链烷磷酸酯、硫代磷酸酯等。上述磷酸酯类似物的制备，以及它们掺入到核苷酸、经修饰的核苷酸和寡核苷酸中本身也为已知的且无需在此描述。In another embodiment, the oligonucleotides comprise nucleic acid sequences set forth in SEQ ID NO: 10-30, including antisense sequences identified and amplified using, eg, PCR, hybridization, and the like. These oligonucleotides may comprise one or more modified nucleotides, shorter or longer fragments, modified linkages, and the like. Examples of modified linkages or internucleotide linkages include phosphorothioate, phosphorodithioate, and the like. In another embodiment, the nucleotides include phosphorus derivatives. Phosphorous derivatives (or modified phosphate groups) that can be attached to sugar or sugar analog moieties in the modified oligonucleotides of the present invention can be monophosphate, diphosphate, triphosphate, alkyl phosphate esters, alkanophosphates, phosphorothioates, etc. The preparation of the abovementioned phosphate analogs, as well as their incorporation into nucleotides, modified nucleotides and oligonucleotides is also known per se and need not be described here.
反义物的特异性和灵敏性也被本领域的技术人员掌握用于治疗用途。已将反义寡核苷酸用作在动物和人的疾病状态治疗中的治疗部分。已将反义寡核苷酸安全和有效地施用给人，并且目前正在进行许多临床试验。因此已确定寡核苷酸可为有用的治疗形式，其可经配置以在用于治疗细胞、组织和动物尤其人的治疗方案中有用。The specificity and sensitivity of antisense for therapeutic use is also within the grasp of those skilled in the art. Antisense oligonucleotides have been used as therapeutic moieties in the treatment of disease states in animals and humans. Antisense oligonucleotides have been safely and effectively administered to humans, and many clinical trials are currently underway. It has thus been determined that oligonucleotides can be useful therapeutic forms that can be formulated to be useful in therapeutic regimens for the treatment of cells, tissues and animals, especially humans.
在本发明的实施方案中，寡聚反义化合物(具体地寡核苷酸)结合靶核酸分子并调节由靶基因编码的分子的表达和/或功能。待干扰的DNA功能包括例如复制和转录。待干扰的RNA功能包括所有的生活机能，例如RNA向蛋白质翻译位点的易位、蛋白质自RNA的翻译、产生一种或更多种mRNA种类的RNA剪接，以及可由RNA参与或促进的催化活性。所述功能可被增量调节或受抑制，这取决于所需的功能。In an embodiment of the invention, oligomeric antisense compounds, in particular oligonucleotides, bind to target nucleic acid molecules and modulate the expression and/or function of the molecule encoded by the target gene. DNA functions to be interfered with include, for example, replication and transcription. RNA functions to be interfered with include all vital functions such as translocation of RNA to protein translation sites, translation of proteins from RNA, RNA splicing to produce one or more mRNA species, and catalytic activities that may be engaged or facilitated by RNA . The function can be incrementally adjusted or inhibited, depending on the desired function.
反义化合物包括反义寡聚化合物、反义寡核苷酸、外部指导序列(EGS)寡核苷酸、可变剪接物、引物、探针和与靶核酸的至少一部分杂交的其它寡聚化合物。因此，这些化合物可以单链、双链、部分单链或环状寡聚化合物的形式引入。Antisense compounds include antisense oligomeric compounds, antisense oligonucleotides, external guide sequence (EGS) oligonucleotides, alternative splices, primers, probes, and other oligomeric compounds that hybridize to at least a portion of a target nucleic acid . Thus, these compounds may be incorporated as single-stranded, double-stranded, partially single-stranded or cyclic oligomeric compounds.
在本发明的情况下，将反义化合物靶向特定的核酸分子可为多步过程。所述过程通常以鉴定待调节其功能的靶核酸开始。此靶核酸可为，例如其表达与特定病症或疾病状态有关的细胞基因(或从基因转录的mRNA)，或来自传染剂的核酸分子。在本发明中，所述靶核酸编码抗病毒基因。In the context of the present invention, targeting an antisense compound to a particular nucleic acid molecule can be a multi-step process. The process generally begins with the identification of a target nucleic acid whose function is to be modulated. Such a target nucleic acid can be, for example, a cellular gene (or mRNA transcribed from a gene) whose expression is associated with a particular disorder or disease state, or a nucleic acid molecule from an infectious agent. In the present invention, the target nucleic acid encodes an antiviral gene.
靶向过程通常也包括确定靶核酸内的至少一个靶区、区段或位点以用于发生反义相互作用，使得产生所需的效应，例如，表达的调节。在本发明的上下文中，术语“区”定义为具有至少一个可识别结构、功能或特征的靶核酸的一部分。靶核酸区内为区段。“区段”定义为在靶核酸内区的较小或亚部分。本发明所用的“位点”定义为靶核酸内的位置。Targeting generally also involves identifying at least one target region, segment or site within a target nucleic acid for antisense interaction to occur such that a desired effect occurs, eg, modulation of expression. In the context of the present invention, the term "region" is defined as a portion of a target nucleic acid having at least one identifiable structure, function or characteristic. Within the region of the target nucleic acid are segments. A "segment" is defined as a smaller or sub-portion of a region within a target nucleic acid. A "site" as used in the present invention is defined as a position within a target nucleic acid.
在一个实施方案中，反义寡核苷酸结合抗病毒基因的天然反义序列，并调节抗病毒基因(SEQ ID NO：1-3)的表达和/或功能。反义序列的实例包括SEQ ID NO：4-30。In one embodiment, the antisense oligonucleotide binds to the natural antisense sequence of the antiviral gene and modulates the expression and/or function of the antiviral gene (SEQ ID NO: 1-3). Examples of antisense sequences include SEQ ID NO: 4-30.
在另一个实施方案中，反义寡核苷酸结合抗病毒基因多核苷酸的一个或更多个区段，并调节抗病毒基因的表达和/或功能。所述区段包含抗病毒基因有义或反义多核苷酸的至少五个连续核苷酸。In another embodiment, an antisense oligonucleotide binds to one or more segments of an antiviral gene polynucleotide and modulates the expression and/or function of the antiviral gene. The segment comprises at least five contiguous nucleotides of the antiviral gene sense or antisense polynucleotide.
在另一个实施方案中，反义寡核苷酸对抗病毒基因的天然反义序列而言为特异性的，其中所述寡核苷酸与抗病毒基因的天然反义序列的结合会调节抗病毒基因的表达和/或功能。In another embodiment, the antisense oligonucleotide is specific for the natural antisense sequence of the antiviral gene, wherein binding of the oligonucleotide to the natural antisense sequence of the antiviral gene modulates the antiviral Gene expression and/or function.
在另一个实施方案中，寡核苷酸化合物包括如SEQ ID NO：10-30所述的序列、使用例如PCR、杂交等鉴定和扩增的反义序列。这些寡核苷酸可包含一个或更多个经修饰的核苷酸、较短或较长的片段、经修饰的键等。经修饰的键或核苷酸间键的实例包括硫代磷酸酯、二硫代磷酸酯等。在另一个实施方案中，所述核苷酸包括磷衍生物。可连接到本发明的修饰的寡核苷酸中的糖或糖类似物部分的磷衍生物(或经修饰的磷酸基)，可为单磷酸酯、二磷酸酯、三磷酸酯、烷基磷酸酯、链烷磷酸酯、硫代磷酸酯等。上述磷酸酯类似物的制备，以及它们掺入到核苷酸、经修饰的核苷酸和寡核苷酸中本身也为已知的且无需在此描述。In another embodiment, the oligonucleotide compound comprises the sequences set forth in SEQ ID NOS: 10-30, antisense sequences identified and amplified using, eg, PCR, hybridization, and the like. These oligonucleotides may comprise one or more modified nucleotides, shorter or longer fragments, modified linkages, and the like. Examples of modified linkages or internucleotide linkages include phosphorothioate, phosphorodithioate, and the like. In another embodiment, the nucleotides include phosphorus derivatives. Phosphorous derivatives (or modified phosphate groups) that can be attached to sugar or sugar analog moieties in the modified oligonucleotides of the present invention can be monophosphate, diphosphate, triphosphate, alkyl phosphate esters, alkanophosphates, phosphorothioates, etc. The preparation of the abovementioned phosphate analogs, as well as their incorporation into nucleotides, modified nucleotides and oligonucleotides is also known per se and need not be described here.
由于如本领域已知，翻译起始密码子通常为5′-AUG(在转录的mRNA分子中；在相应的DNA分子中为5′-ATG)，因而翻译起始密码子也称为“AUG密码子”、“起始密码子”或“AUG起始密码子”。少数基因具有翻译起始密码子，其具有RNA序列5′-GUG、5′-UUG或5′-CUG；以及5′-AUA、5′-ACG和5′-CUG已显示在体内起作用。因此，术语“翻译起始密码子”和“起始密码子”可包括许多密码子序列，但在每个情况下起始氨基酸通常为甲硫氨酸(在真核生物中)或甲酰甲硫氨酸(在原核生物中)。真核和原核基因可具有两个或更多个备选起始密码子，其中的任何一个可优先地用于在特定细胞类型或组织中或在特定条件组下的翻译起始。在本发明的上下文中，“起始密码子”和“翻译起始密码子”是指这样的一个或复数个密码子，其在体内用于起始由编码抗病毒基因的基因转录的mRNA的翻译，与这类密码子的序列无关。基因的翻译终止密码子(或“终止密码子”)可具有三个序列中的一个，即，5′-UAA、5′-UAG和5′-UGA(对应的DNA序列分别为5′-TAA、5′-TAG和5′-TGA)。Since, as is known in the art, the translation initiation codon is usually 5'-AUG (in transcribed mRNA molecules; 5'-ATG in corresponding DNA molecules), the translation initiation codon is also referred to as "AUG codon", "start codon" or "AUG start codon". A few genes have translation initiation codons with the RNA sequences 5'-GUG, 5'-UUG or 5'-CUG; and 5'-AUA, 5'-ACG and 5'-CUG have been shown to function in vivo. Thus, the terms "translation initiation codon" and "initiation codon" may include a number of codon sequences, but in each case the starting amino acid is usually methionine (in eukaryotes) or formylform Thionine (in prokaryotes). Eukaryotic and prokaryotic genes may have two or more alternative initiation codons, any of which may be preferentially used for translation initiation in a particular cell type or tissue or under a particular set of conditions. In the context of the present invention, "initiation codon" and "translation initiation codon" refer to the codon or codons used in vivo to initiate the transcription of mRNA transcribed from a gene encoding an antiviral gene. Translation is independent of the sequence of such codons. The translation termination codon (or "stop codon") of a gene can have one of three sequences, namely, 5'-UAA, 5'-UAG, and 5'-UGA (the corresponding DNA sequence is 5'-TAA , 5'-TAG and 5'-TGA).
术语“起始密码子区”和“翻译起始密码子区”是指从翻译起始密码子开始在任一方向上(即，5’或3’)包含约25-约50个连续的核苷酸的这类mRNA或基因的部分。类似地，术语“终止密码子区”和“翻译终止密码子区”是指从翻译终止密码子开始在任一方向上(即，5’或3’)包含约25-约50个连续的核苷酸的这类mRNA或基因的部分。因此，“起始密码子区”(或“翻译起始密码子区”)和“终止密码子区”(或“翻译终止密码子区”)均为可用本发明的反义化合物有效地靶向的区。The terms "initiation codon region" and "translation initiation codon region" refer to a region comprising about 25 to about 50 contiguous nucleotides in either direction (i.e., 5' or 3') from the translation initiation codon Portions of such mRNA or genes. Similarly, the terms "stop codon region" and "translation stop codon region" refer to a region comprising about 25 to about 50 contiguous nucleotides in either direction (i.e., 5' or 3') from the translation stop codon Portions of such mRNA or genes. Thus, both the "initiation codon region" (or "translation initiation codon region") and the "termination codon region" (or "translation stop codon region") are effectively targetable with the antisense compounds of the invention. area.
本领域已知的开放阅读框(ORF)或“编码区”是指在翻译起始密码子和翻译终止密码子之间的区，也为可有效地靶向的区。在本发明的上下文内，靶向的区为包含基因的开放阅读框(ORF)的翻译起始或终止密码子的基因内区。An open reading frame (ORF) or "coding region" as known in the art refers to the region between the translation initiation codon and the translation termination codon, which is also effectively targetable. Within the context of the present invention, the targeted region is the intragenic region comprising the translation initiation or termination codon of the open reading frame (ORF) of the gene.
另一种靶区包括本领域已知的5’非翻译区(5’UTR)，是指在翻译起始密码子的5’方向上的mRNA的部分，因此包括在mRNA的5’加帽位点和翻译起始密码子之间的核苷酸(或基因上对应的核苷酸)。再一种靶区包括本领域已知的3’非翻译区(3′UTR)，是指在翻译终止密码子3’方向上的mRNA的部分，因此包括在mRNA的翻译终止密码子和3’末端之间的核苷酸(或基因上对应的核苷酸)。mRNA的5’加帽位点包含经由5’-5’三磷酸酯键连接到mRNA的5’最末端残基的N7-甲基化鸟苷残基。认为mRNA的5’帽区包括5’帽子结构本身以及邻近该加帽位点的前50个核苷酸。用于本发明的另一种靶区为5’帽区。Another target region includes what is known in the art as the 5' untranslated region (5'UTR), which refers to the portion of the mRNA in the 5' direction of the translation initiation codon, thus including the 5' capping position of the mRNA The nucleotides between the dot and the translation initiation codon (or the corresponding nucleotides on the gene). Yet another target region includes the 3' untranslated region (3'UTR) known in the art, which refers to the portion of the mRNA in the 3' direction of the translation stop codon, thus including the translation stop codon and the 3' The nucleotides between the ends (or the corresponding nucleotides on the gene). The 5' capping site of the mRNA contains an N7-methylated guanosine residue linked to the 5'-most residue of the mRNA via a 5'-5' triphosphate bond. The 5' cap region of an mRNA is considered to include the 5' cap structure itself and the first 50 nucleotides adjacent to the capping site. Another target region for use in the present invention is the 5' cap region.
尽管一些真核mRNA转录物为直接翻译的，但是许多包含一个或更多个称为“内含子”的区，其在翻译前被从转录物中切除。余下的(且因此翻译的)区称为“外显子”，并将其剪接在一起形成连续的mRNA序列。在一个实施方案中，靶向剪接位点(即，内含子-外显子连接处或外显子-内含子连接处)在疾病牵涉异常剪接或疾病牵涉特定剪接产物过度产生的状况中特别有用。因重排或缺失所致的异常融合连接处为靶位点的另一个实施方案。经由来自不同基因来源的两个(或更多个)mRNA的剪接过程产生的mRNA转录物称为“融合转录物”。内含子可使用靶向例如DNA或前-mRNA的反义化合物来有效地靶向。Although some eukaryotic mRNA transcripts are directly translated, many contain one or more regions called "introns," which are excised from the transcript prior to translation. The remaining (and thus translated) regions are called "exons" and are spliced together to form a continuous mRNA sequence. In one embodiment, splice sites (i.e., intron-exon junctions or exon-intron junctions) are targeted in conditions where aberrant splicing is involved in a disease or the overproduction of a particular splice product is involved in a disease Very useful. Aberrant fusion junctions due to rearrangements or deletions are another embodiment of the target site. An mRNA transcript produced via the splicing process of two (or more) mRNAs from different gene sources is called a "fusion transcript". Introns can be effectively targeted using antisense compounds that target, for example, DNA or pre-mRNA.
在另一个实施方案中，反义寡核苷酸结合靶多核苷酸的编码和/或非编码区，并调节靶分子的表达和/或功能。In another embodiment, an antisense oligonucleotide binds to coding and/or non-coding regions of a target polynucleotide and modulates the expression and/or function of the target molecule.
在另一个实施方案中，反义寡核苷酸结合天然反义多核苷酸，并调节靶分子的表达和/或功能。In another embodiment, the antisense oligonucleotides bind natural antisense polynucleotides and modulate the expression and/or function of the target molecule.
在另一个实施方案中，反义寡核苷酸结合有义多核苷酸，并调节靶分子的表达和/或功能。In another embodiment, an antisense oligonucleotide binds a sense polynucleotide and modulates the expression and/or function of a target molecule.
可变RNA转录物可产生自DNA的相同基因组区。这些可变转录物一般称为“变体”。更具体地，“前mRNA变体”为产生自相同的基因组DNA的转录物，其与产生自相同的基因组DNA的其它转录物在其起始或终止位置上不同且包含内含子和外显子序列二者。Alternative RNA transcripts can arise from the same genomic region of DNA. These alternative transcripts are generally referred to as "variants". More specifically, a "pre-mRNA variant" is a transcript produced from the same genomic DNA that differs in its start or end position from other transcripts produced from the same genomic DNA and that contains introns and exons. Both subsequences.
当剪接期间切除了一个或更多个外显子或内含子区、或其部分时，前mRNA变体产生更小的“mRNA变体”。因此，mRNA变体为经加工的前mRNA变体，并且由于剪接所致，每种独特的前mRNA变体必须总是产生独特的mRNA变体。这些mRNA变体也称为“可变剪接变体”。如果未发生前mRNA变体的剪接，则前mRNA变体与mRNA变体完全相同。Pre-mRNA variants produce smaller "mRNA variants" when one or more exonic or intronic regions, or portions thereof, are excised during splicing. Thus, mRNA variants are processed pre-mRNA variants, and each unique pre-mRNA variant must always result in a unique mRNA variant due to splicing. These mRNA variants are also referred to as "alternative splice variants". A pre-mRNA variant is identical to an mRNA variant if no splicing of the pre-mRNA variant occurs.
变体可通过使用可变信号启动或终止转录来产生。前mRNA和mRNA可具有多于一个起始密码子或终止密码子。起源于使用可变起始密码子的前mRNA或mRNA的变体称为该前mRNA或mRNA的“可变起始变体”。使用可变终止密码子的转录物称为该前mRNA或mRNA的“可变终止变体”。可变终止变体的一个具体类型为“聚腺苷酸变体”，其中所产生的多重转录物起因于转录机制对其中一种“聚腺苷酸终止信号”的可变选择，从而产生终止在独特的聚腺苷酸位点上的转录物。在本发明的上下文内，本文所述的变体类型也为靶核酸的实施方案。Variants can be produced by using alternative signals to initiate or terminate transcription. Pre-mRNA and mRNA can have more than one start codon or stop codon. Variants originating from a pre-mRNA or mRNA that use an alternative start codon are referred to as "alternative start variants" of that pre-mRNA or mRNA. Transcripts using alternative stop codons are referred to as "alternative stop variants" of that pre-mRNA or mRNA. A specific type of variable termination variant is the "polyA variant", in which multiple transcripts are produced resulting from the variable selection of one of the "polyA termination signals" by the transcription machinery, resulting in termination Transcripts at unique polyA sites. Within the context of the present invention, the variant types described herein are also embodiments of the target nucleic acid.
将反义化合物与之杂交的靶核酸上的位置定义为活性反义化合物靶向的靶区的至少5个核苷酸长的部分。The position on the target nucleic acid to which the antisense compound hybridizes is defined as the portion at least 5 nucleotides long of the target region to which the active antisense compound is targeted.
虽然将某些示例性靶区段的具体序列列举于此，但是本领域的技术人员会认识到，这些用于说明和描述在本发明范围内的具体实施方案。根据本公开内容，其它靶区段可由本领域普通技术人员容易地鉴定。While specific sequences of certain exemplary target segments are set forth herein, those skilled in the art will recognize that these are for illustration and description of specific embodiments within the scope of the invention. Other target segments can be readily identified by one of ordinary skill in the art in light of the present disclosure.
认为以下靶区段同样适合靶向，该靶区段长度为5-100个核苷酸并包含选自说明性的靶区段之内的至少五(5)个连续核苷酸的片段。Target segments that are 5-100 nucleotides in length and that comprise a stretch of at least five (5) contiguous nucleotides selected from within the illustrative target segments are also considered suitable for targeting.
靶区段可包括DNA或RNA序列，其包含来自说明性靶区段之一的5’末端的至少5个连续核苷酸(余下的核苷酸为相同DNA或RNA的连续片段，其开始于靶区段5’末端的紧接上游且持续到该DNA或RNA包含约5-约100个核苷酸为止)。类似地，靶区段由以下DNA或RNA序列表示，该序列包含来自说明性靶区段之一的3’末端的至少5个连续核苷酸(余下的核苷酸为相同DNA或RNA的连续段，其开始于靶区段3’末端的紧接下游且持续到该DNA或RNA包含约5-约100个核苷酸为止)。本领域技术人员根据本文所说明的靶区段，无需过度试验就能够鉴定其它靶区段。A target segment may comprise a DNA or RNA sequence comprising at least 5 contiguous nucleotides from the 5' end of one of the illustrative target segments (the remaining nucleotides being a contiguous stretch of the same DNA or RNA that begins at immediately upstream of the 5' end of the target segment and continues until the DNA or RNA comprises about 5 to about 100 nucleotides). Similarly, a target segment is represented by a DNA or RNA sequence comprising at least 5 contiguous nucleotides from the 3' end of one of the illustrative target segments (the remaining nucleotides are contiguous nucleotides of the same DNA or RNA). segment that begins immediately downstream of the 3' end of the target segment and continues until the DNA or RNA comprises about 5 to about 100 nucleotides). Based on the target segments described herein, one skilled in the art will be able to identify other target segments without undue experimentation.
一旦鉴定一个或更多个靶区、区段或位点，就选出与该靶足够互补的反义化合物，所述足够互补即充分良好且具有足够的特异性地杂交以得到所需的效果。Once one or more target regions, segments or sites are identified, antisense compounds are selected that are sufficiently complementary to that target to hybridize well enough and with sufficient specificity to have the desired effect .
在本发明的实施方案中，寡核苷酸与特定靶标的反义链结合。所述寡核苷酸长度为至少5个核苷酸且可为合成的，使得每个寡核苷酸靶向重叠的序列，由此将寡核苷酸合成为覆盖靶多核苷酸的全长。靶标也包括编码区以及非编码区。In an embodiment of the invention, the oligonucleotide binds to the antisense strand of a specific target. The oligonucleotides are at least 5 nucleotides in length and may be synthesized such that each oligonucleotide targets overlapping sequences whereby the oligonucleotides are synthesized to cover the full length of the target polynucleotide . Targets also include coding as well as non-coding regions.
在一个实施方案中，通过反义寡核苷酸来靶向特定核酸。将反义化合物靶向特定核酸为多步过程。该过程通常开始于鉴定其功能待调节的核酸序列。这可为，例如其表达与特定的病症或疾病状态有关的细胞基因(或从该基因转录的mRNA)，或非编码多核苷酸，例如非编码RNA(ncRNA)。In one embodiment, specific nucleic acids are targeted by antisense oligonucleotides. Targeting antisense compounds to specific nucleic acids is a multi-step process. The process generally begins with the identification of a nucleic acid sequence whose function is to be modulated. This could be, for example, a cellular gene (or mRNA transcribed from the gene) whose expression is associated with a particular disorder or disease state, or a non-coding polynucleotide, such as a non-coding RNA (ncRNA).
可将RNA归类为(1)信使RNA(mRNA)，其被翻译成蛋白，和(2)非编码蛋白的RNA(ncRNA)。ncRNA包括微小RNA、反义转录物和包含高密度的终止密码子并缺少任何广泛的“开放阅读框”的其它转录单元(TU)。许多ncRNA似乎开始于蛋白编码基因座的3’非翻译区(3′UTR)中的起始位点。ncRNA常常为罕见的且至少一半已由FANTOM协会测序的ncRNA似乎未聚腺苷酸化。大多数研究者因为明显的原因而关注经加工并输出到细胞质的聚腺苷酸化mRNA。近来，已显示非聚腺苷酸化核RNA的群体可非常巨大，且许多这类转录物产生于基因间区。ncRNA调节基因表达的机制可为通过与靶转录物的碱基配对。通过碱基配对起作用的RNA可分组成(1)顺式编码RNA，其在相同的基因位置、但在其所作用的RNA相反的链上编码，因此显示对其靶标完美的互补性，和(2)反式编码RNA，其在与其所作用的RNA不同的染色体位置上编码，一般不表现出与其靶标完美的碱基配对潜能。RNA can be categorized as (1) messenger RNA (mRNA), which is translated into protein, and (2) non-protein-coding RNA (ncRNA). ncRNAs include microRNAs, antisense transcripts, and other transcription units (TUs) that contain a high density of stop codons and lack any extensive "open reading frames." Many ncRNAs appear to start at initiation sites in the 3' untranslated regions (3' UTRs) of protein-coding loci. ncRNAs are often rare and at least half of the ncRNAs sequenced by the FANTOM consortium do not appear to be polyadenylated. Most investigators focus on polyadenylated mRNAs that are processed and exported to the cytoplasm for obvious reasons. Recently, it has been shown that the population of non-polyadenylated nuclear RNAs can be very large and that many such transcripts are produced in intergenic regions. The mechanism by which ncRNAs regulate gene expression may be through base pairing with target transcripts. RNAs that act by base pairing can be grouped into (1) cis-coding RNAs, which are encoded at the same genetic position but on the opposite strand of the RNA they act on and thus exhibit perfect complementarity to their targets, and (2) Trans-coding RNAs, which are encoded at a different chromosomal location than the RNA they act on, generally do not exhibit perfect base-pairing potential with their targets.
不希望受到理论的约束，通过本文所述的反义寡核苷酸来扰乱反义多核苷酸，可改变相应有义信使RNA的表达。然而，此调节可为不一致的(反义敲减导致信使RNA上升)或一致的(反义敲减导致伴随的信使RNA下降)。在这些情况下，可将反义寡核苷酸靶向反义转录物的重叠或非重叠部分，引起其敲减或隔离。编码以及非编码反义物可以相同的方式来靶向，并且任一种类别均能够调节相应有义转录物——以一致或不一致的方式。用于鉴定针对靶标使用的新寡核苷酸的策略可基于通过反义寡核苷酸或任何其它调节所需靶标的方法来敲减反义RNA转录物。Without wishing to be bound by theory, disruption of an antisense polynucleotide by an antisense oligonucleotide as described herein alters the expression of the corresponding sense messenger RNA. However, this regulation can be inconsistent (antisense knockdown results in an increase in messenger RNA) or consistent (antisense knockdown results in a concomitant decrease in messenger RNA). In these cases, antisense oligonucleotides can be targeted to overlapping or non-overlapping portions of antisense transcripts, causing their knockdown or sequestration. Coding as well as non-coding antisenses can be targeted in the same manner, and either class can modulate the corresponding sense transcript—consistently or discordantly. A strategy for identifying new oligonucleotides for use against a target can be based on knockdown of antisense RNA transcripts by antisense oligonucleotides or any other method of modulating the desired target.
策略1：在不一致调节的情况下，敲减所述反义转录物提升常规(有义)基因的表达。若后者基因编码已知或假定的药物靶标，则其反义对应物的敲减可预料到地模拟受体激动剂或酶刺激剂的作用。Strategy 1: Knockdown of the antisense transcript elevates the expression of the canonical (sense) gene in the case of discordant regulation. If the latter gene encodes a known or putative drug target, knockdown of its antisense counterpart predictably mimics the effect of a receptor agonist or enzyme stimulator.
策略2：在一致调节的情况下，可伴随地敲减反义和有义转录物两者，从而达到常规(有义)基因表达的协同下降。如果例如将反义寡核苷酸用于进行敲减，则此策略可用于应用针对有义转录物靶向的一种反义寡核苷酸和针对相应反义转录物的另一种反义寡核苷酸，或同时靶向重叠的有义和反义转录物的单个有力对称的反义寡核苷酸。Strategy 2: In the case of concerted regulation, both antisense and sense transcripts can be knocked down concomitantly, thereby achieving a synergistic decrease in canonical (sense) gene expression. If for example antisense oligonucleotides are used to perform the knockdown, this strategy can be used to apply one antisense oligonucleotide targeted to the sense transcript and another antisense to the corresponding antisense transcript oligonucleotides, or a single potent symmetric antisense oligonucleotide that simultaneously targets overlapping sense and antisense transcripts.
根据本发明，反义化合物包括反义寡核苷酸、核酶、外部指导序列(EGS)寡核苷酸、siRNA化合物、单链或双链RNA干扰(RNAi)化合物(例如siRNA化合物)，以及与靶核酸的至少一部分杂交且调节其功能的其它寡聚化合物。因此，其可为DNA、RNA、DNA样、RNA样、或其混合物，或可为这些中的一种或更多种的模拟物。这些化合物可为单链、双链、环状或发夹寡聚化合物且可包含结构元件，例如内部或末端突起、错配或环。将反义化合物常规地制备为线性的但可被连接，或者另外制备成环状和/或分枝的。反义化合物可包括构建体，例如杂交以形成完全或部分双链化合物的两条链，或具有足够自我互补性以允许杂交并形成完全或部分双链化合物的单链。可将所述两条链内部连接而留下自由的3’或5’末端，或可将其连接形成连续的发夹结构或环。发夹结构可在5’或3’末端上包含突出端，产生单链特征的延伸。所述双链化合物任选可在末端上包含突出端。进一步的修饰可包括与末端之一、经挑选的核苷酸位置、糖位置或与核苷酸间键之一连接的缀合基团。或者，所述两条链可经由非核酸部分或连接基团来连接。当仅由一条链形成时，dsRNA可呈自我互补的发夹型分子形式，其在其自身上对折形成双链体。因此，所述dsRNA可为完全或部分双链的。基因表达的特异性调节可通过在转基因细胞系中稳定表达dsRNA发夹来完成，然而，在一些实施方案中，基因表达或功能为增量调节的。当形成自两条链，或呈在其自身上对折形成双链体的自身互补发夹型分子形式的单链时，所述两条链(或单链的双链体形成区)为以沃森-克里克模式碱基配对的互补RNA链。According to the present invention, antisense compounds include antisense oligonucleotides, ribozymes, external guide sequence (EGS) oligonucleotides, siRNA compounds, single- or double-stranded RNA interference (RNAi) compounds (such as siRNA compounds), and Other oligomeric compounds that hybridize to at least a portion of a target nucleic acid and modulate its function. Thus, it may be DNA, RNA, DNA-like, RNA-like, or a mixture thereof, or may be a mimetic of one or more of these. These compounds may be single-stranded, double-stranded, circular or hairpin oligomeric compounds and may contain structural elements such as internal or terminal protrusions, mismatches or loops. Antisense compounds are conventionally prepared as linear but may be linked, or otherwise made circular and/or branched. Antisense compounds may include constructs, such as two strands that hybridize to form a fully or partially double-stranded compound, or a single strand that is sufficiently self-complementary to allow hybridization and form a fully or partially double-stranded compound. The two strands can be joined internally leaving the 3' or 5' ends free, or they can be linked to form a continuous hairpin structure or loop. The hairpin structure may contain an overhang on the 5' or 3' end, creating an extension that is characteristic of a single strand. The double stranded compound optionally may comprise overhangs on the ends. Further modifications may include a conjugation group attached to one of the termini, selected nucleotide positions, sugar positions or to one of the internucleotide linkages. Alternatively, the two strands may be linked via a non-nucleic acid moiety or linking group. When formed from only one strand, dsRNA can be in the form of a self-complementary hairpin-type molecule that folds on itself to form a duplex. Thus, the dsRNA can be fully or partially double-stranded. Specific regulation of gene expression can be accomplished by stably expressing dsRNA hairpins in transgenic cell lines, however, in some embodiments, gene expression or function is upregulated. When formed from two strands, or a single strand in the form of a self-complementary hairpin molecule that folds on itself to form a duplex, the two strands (or the duplex-forming region of the single strand) are Complementary RNA strands that are base-paired in the Sen-Crick pattern.
一旦引入系统，本发明的化合物可引起一种或更多种酶或结构蛋白的作用以实现靶核酸的切割或其它修饰，或可经由基于占据的机制来运作。一般而言，核酸(包括寡核苷酸)可描述为“DNA样”(即，一般具有一个或更多个2’脱氧糖和一般地T而不是U碱基)或“RNA样”(即，一般具有一个或更多个2’羟基或2’修饰的糖和一般U而不是T碱基)。核酸螺旋可采取多于一种类型的结构，最普通地A和B型。据认为，一般而言，具有B型样结构的寡核苷酸为“DNA样”而具有A型样结构的寡核苷酸为“RNA样”。在一些(嵌合的)实施方案中，反义化合物可包含A和B型区两者。Once introduced into the system, the compounds of the invention may cause the action of one or more enzymes or structural proteins to effect cleavage or other modification of the target nucleic acid, or may operate via an occupancy-based mechanism. In general, nucleic acids (including oligonucleotides) can be described as "DNA-like" (i.e., generally having one or more 2' deoxysugars and generally T rather than U bases) or "RNA-like" (i.e. , generally with one or more 2' hydroxyl or 2' modified sugars and generally U rather than T bases). Nucleic acid helices can adopt more than one type of structure, most commonly A and B types. In general, oligonucleotides with a B-like structure are considered "DNA-like" and oligonucleotides with an A-like structure are "RNA-like". In some (chimeric) embodiments, an antisense compound may comprise both A and B-type regions.
在另一个实施方案中，所需的寡核苷酸或反义化合物，包括以下中的至少一种：反义RNA、反义DNA、嵌合反义寡核苷酸、包含经修饰的键的反义寡核苷酸、干扰RNA(RNAi)、短干扰RNA(siRNA)；微小干扰RNA(miRNA)；小时序RNA(stRNA)；或短发夹RNA(shRNA)；小RNA诱导的基因激活(RNAa)；小激活RNA(saRNA)、或其组合。In another embodiment, the desired oligonucleotide or antisense compound comprises at least one of the following: antisense RNA, antisense DNA, chimeric antisense oligonucleotides, Antisense oligonucleotides, interfering RNA (RNAi), short interfering RNA (siRNA); micro interfering RNA (miRNA); small sequential RNA (stRNA); or short hairpin RNA (shRNA); small RNA-induced gene activation ( RNAa); small activating RNA (saRNA), or a combination thereof.
dsRNA也可激活基因表达，这是已被称为“小RNA诱导的基因激活”或RNAa的机制。靶向基因启动子的dsRNA诱导相关基因的有效转录激活。在人细胞中使用合成dsRNA(称为“小激活RNA”(saRNA))证实RNAa。dsRNA can also activate gene expression, a mechanism that has been termed "small RNA-induced gene activation," or RNAa. dsRNAs targeting gene promoters induce efficient transcriptional activation of associated genes. RNAa was demonstrated in human cells using synthetic dsRNA, termed "small activating RNA" (saRNA).
已发现小双链RNA(dsRNA)(例如小干扰RNA(siRNA)和微小RNA(miRNA))是称为RNA干扰(RNAi)的进化保守机制的触发物。RNAi总是导致基因沉默。然而，在下文实施例章节详述的例子中，显示寡核苷酸会增加抗病毒基因多核苷酸和其编码产物的表达和/或功能。dsRNA也可充当小激活RNA(saRNA)。不希望受理论约束，通过靶向基因启动子中的序列，saRNA在称为dsRNA诱导的转录激活(RNAa)的现象中诱导靶基因表达。Small double-stranded RNAs (dsRNAs), such as small interfering RNAs (siRNAs) and microRNAs (miRNAs), have been found to be triggers of an evolutionarily conserved mechanism known as RNA interference (RNAi). RNAi always results in gene silencing. However, in the examples detailed in the Examples section below, oligonucleotides were shown to increase the expression and/or function of antiviral gene polynucleotides and their encoded products. dsRNA can also act as small activating RNA (saRNA). Without wishing to be bound by theory, by targeting sequences in gene promoters, saRNA induces target gene expression in a phenomenon known as dsRNA-induced transcriptional activation (RNAa).
在另一个实施方案中，本文鉴定的“靶区段”可用于筛选调节抗病毒基因多核苷酸表达的另外化合物。“调节剂”为这样的化合物：其减少或增加编码抗病毒基因的核酸分子的表达，并至少包含与靶区段互补的5-核苷酸部分。筛选方法包括以下步骤：使编码抗病毒基因的有义或天然反义多核苷酸的核酸分子的靶区段与一种或更多种候选调节剂接触，以及选择一种或更多种减少或增加编码抗病毒基因多核苷酸(例如SEQ ID NO：10-30)的核酸分子表达的候选调节剂。一旦证实一种或更多种候选调节剂能够调节(例如减少或增加)编码抗病毒基因多核苷酸的核酸分子表达，则可将所述调节剂用于抗病毒基因多核苷酸功能的进一步调查研究，或用作根据本发明的研究、诊断或治疗剂。In another embodiment, the "target segments" identified herein can be used to screen for additional compounds that modulate the expression of antiviral gene polynucleotides. A "modulator" is a compound that reduces or increases the expression of a nucleic acid molecule encoding an antiviral gene and that comprises at least a 5-nucleotide portion that is complementary to a target segment. The screening method comprises the steps of contacting a target segment of a nucleic acid molecule encoding a sense or natural antisense polynucleotide encoding an antiviral gene with one or more candidate modulators, and selecting one or more that reduce or Candidate modulators that increase expression of nucleic acid molecules encoding antiviral gene polynucleotides (eg, SEQ ID NO: 10-30). Once it is confirmed that one or more candidate modulators are capable of modulating (e.g., decreasing or increasing) the expression of a nucleic acid molecule encoding an antiviral gene polynucleotide, the modulators can be used for further investigation of the function of the antiviral gene polynucleotide research, or use as a research, diagnostic or therapeutic agent according to the invention.
靶向天然反义序列会调节靶基因的功能。例如，抗病毒基因(例如登录号NM_014314、NM_022168、NM_024013)。在一个实施方案中，靶标为抗病毒基因的反义多核苷酸。在一个实施方案中，反义寡核苷酸靶向抗病毒基因多核苷酸(例如登录号NM_014314、NM_022168、NM_024013)的有义和/或天然反义序列、其变体、等位基因、同种型、同源物、突变体、衍生物、片段和互补序列。优选地，所述寡核苷酸为反义分子，且所述靶标包括反义和/或有义抗病毒基因多核苷酸的编码区和非编码区。Targeting natural antisense sequences modulates the function of the target gene. For example, antiviral genes (eg accession numbers NM_014314, NM_022168, NM_024013). In one embodiment, the target is an antisense polynucleotide of an antiviral gene. In one embodiment, the antisense oligonucleotides target the sense and/or natural antisense sequences of antiviral gene polynucleotides (e.g. accession numbers NM_014314, NM_022168, NM_024013), variants, alleles, isomorphic Isoforms, homologues, mutants, derivatives, fragments and complementary sequences. Preferably, the oligonucleotide is an antisense molecule, and the target includes the coding region and non-coding region of the antisense and/or sense antiviral gene polynucleotide.
本发明的靶区段也可与本发明的其各自互补的反义化合物结合，以形成稳定的双链(双链体)寡核苷酸。Target segments of the invention may also be combined with their respective complementary antisense compounds of the invention to form stable double-stranded (duplex) oligonucleotides.
本领域中已显示这类双链寡核苷酸部分经由反义机制来调节靶表达和调节翻译以及RNA加工。此外，所述双链部分可经受化学修饰。例如，已显示这类双链部分通过所述双链体的反义链与靶标的典型杂交来抑制该靶标，从而触发靶标的酶促降解。Such double-stranded oligonucleotide moieties have been shown in the art to modulate target expression and regulate translation and RNA processing via an antisense mechanism. In addition, the double-stranded portion can be chemically modified. For example, such double-stranded moieties have been shown to inhibit the target through typical hybridization of the antisense strand of the duplex to the target, thereby triggering enzymatic degradation of the target.
在一个实施方案中，反义寡核苷酸靶向抗病毒基因多核苷酸(例如登录号NM_014314、NM_022168、NM_024013)、其变体、等位基因、同种型、同源物、突变体、衍生物、片段和互补序列。优选地，所述寡核苷酸为反义分子。In one embodiment, the antisense oligonucleotides target antiviral gene polynucleotides (e.g. accession numbers NM_014314, NM_022168, NM_024013), variants, alleles, isoforms, homologues, mutants thereof, Derivatives, fragments and complementary sequences. Preferably, said oligonucleotide is an antisense molecule.
根据本发明的实施方案，靶核酸分子不限于单独的抗病毒基因，而是延伸到抗病毒基因分子的任何同种型、受体、同源物等等。According to an embodiment of the present invention, the target nucleic acid molecule is not limited to a single antiviral gene, but extends to any isotype, receptor, homologue, etc. of an antiviral gene molecule.
在另一个实施方案中，寡核苷酸靶向抗病毒基因多核苷酸的天然反义序列，例如，如SEQ ID NO：4-9所述的多核苷酸以及其任何变体、等位基因、同源物、突变体、衍生物、片段和互补序列。反义寡核苷酸的实例如SEQ ID NO：10-30所述。In another embodiment, the oligonucleotide targets the natural antisense sequence of an antiviral gene polynucleotide, for example, a polynucleotide as set forth in SEQ ID NO: 4-9 and any variants, alleles thereof , homologues, mutants, derivatives, fragments and complementary sequences. Examples of antisense oligonucleotides are set forth in SEQ ID NO: 10-30.
在一个实施方案中，所述寡核苷酸与抗病毒基因反义物的核酸序列互补或结合，并调节抗病毒基因分子的表达和/或功能，所述核酸序列包括但不限于与抗病毒基因多核苷酸有关的非编码有义和/或反义序列。In one embodiment, the oligonucleotide is complementary or combined with the nucleic acid sequence of the antiviral gene antisense, and regulates the expression and/or function of the antiviral gene molecule, and the nucleic acid sequence includes but is not limited to antiviral gene antisense Non-coding sense and/or antisense sequences related to gene polynucleotides.
在另一个实施方案中，所述寡核苷酸与如SEQ ID NO：4-9所述的抗病毒基因天然反义物的核酸序列互补或结合，并调节抗病毒基因分子的表达和/或功能。In another embodiment, the oligonucleotide is complementary or combined with the nucleic acid sequence of the antiviral gene natural antisense as described in SEQ ID NO: 4-9, and regulates the expression of the antiviral gene molecule and/or Function.
在一个实施方案中，寡核苷酸包含SEQ ID NO：10-30的至少5个连续核苷酸的序列，且调节抗病毒基因分子的表达和/或功能。In one embodiment, the oligonucleotide comprises a sequence of at least 5 contiguous nucleotides of SEQ ID NO: 10-30, and modulates the expression and/or function of an antiviral gene molecule.
多核苷酸靶标包括抗病毒基因，包括其家族成员、抗病毒基因的变体；抗病毒基因的突变体，包括SNP；抗病毒基因的非编码序列；抗病毒基因的等位基因；物种变体、片段等等。优选地，所述寡核苷酸为反义分子。Polynucleotide targets include antiviral genes, including their family members, variants of antiviral genes; mutants of antiviral genes, including SNPs; non-coding sequences of antiviral genes; alleles of antiviral genes; species variants , fragments, and so on. Preferably, said oligonucleotide is an antisense molecule.
在另一个实施方案中，靶向抗病毒基因多核苷酸的寡核苷酸包括：反义RNA、干扰RNA(RNAi)、短干扰RNA(siRNA)；微小干扰RNA(miRNA)；小时序RNA(stRNA)；或短发夹RNA(shRNA)；小RNA诱导的基因激活(RNAa)；或小激活RNA(saRNA)。In another embodiment, the oligonucleotides targeting antiviral gene polynucleotides include: antisense RNA, interfering RNA (RNAi), short interfering RNA (siRNA); micro interfering RNA (miRNA); small sequential RNA ( stRNA); or short hairpin RNA (shRNA); small RNA-induced gene activation (RNAa); or small activating RNA (saRNA).
在另一个实施方案中，抗病毒基因多核苷酸(例如SEQ ID NO：4-9)的靶向会调节这些靶标的表达或功能。在一个实施方案中，表达或功能相比于对照为增量调节的。在另一个实施方案中，表达或功能相比于对照为减量调节的。In another embodiment, targeting of antiviral gene polynucleotides (eg, SEQ ID NO: 4-9) modulates the expression or function of these targets. In one embodiment, expression or function is upregulated compared to a control. In another embodiment, expression or function is down-regulated compared to a control.
在另一个实施方案中，反义化合物包括如SEQ ID NO：10-30所述的序列。这些寡核苷酸可包含一个或更多个经修饰的核苷酸、更短或更长的片段、经修饰的键等等。In another embodiment, the antisense compound comprises the sequence set forth in SEQ ID NO: 10-30. These oligonucleotides may contain one or more modified nucleotides, shorter or longer fragments, modified linkages, and the like.
在另一个实施方案中，SEQ ID NO：10-30包含一个或更多个LNA核苷酸。In another embodiment, SEQ ID NOs: 10-30 comprise one or more LNA nucleotides.
所需靶核酸的调节可以本领域已知的数个方式来进行。例如，反义寡核苷酸、siRNA等。酶促核酸分子(例如，核酶)为能够催化一种或更多种不同反应(包括以核苷酸碱基序列特异性的方式反复切割其它单独的核酸分子的能力)的核酸分子。这类酶促核酸分子可用于，例如，靶向几乎任何RNA转录物。Modulation of a desired target nucleic acid can be performed in several ways known in the art. For example, antisense oligonucleotides, siRNA, etc. Enzymatic nucleic acid molecules (eg, ribozymes) are nucleic acid molecules capable of catalyzing one or more distinct reactions, including the ability to repeatedly cleave other individual nucleic acid molecules in a nucleotide base sequence-specific manner. Such enzymatic nucleic acid molecules can be used, for example, to target virtually any RNA transcript.
由于反式切割酶促核酸分子的序列特异性，其有望作为用于人类疾病的治疗剂。可将酶促核酸分子设计成在细胞RNA背景下切割特定的RNA靶标。这类切割事件致使mRNA无功能且终止从该RNA的蛋白表达。以这种方式，可选择性地抑制与疾病状态有关的蛋白合成。Due to the sequence specificity of trans-cleaving enzymatic nucleic acid molecules, they hold promise as therapeutic agents for human diseases. Enzymatic nucleic acid molecules can be designed to cleave specific RNA targets in the context of cellular RNA. Such cleavage events render the mRNA nonfunctional and terminate protein expression from that RNA. In this way, protein synthesis associated with disease states can be selectively inhibited.
一般而言，带有RNA切割活性的酶促核酸通过首先与靶RNA结合来起作用。这类结合通过酶促核酸的靶结合部分来进行，该酶促核酸的靶结合部分保持紧密靠近进行切割靶RNA的分子的酶促部分。因此，所述酶促核酸首先识别而后通过互补的碱基配对与靶RNA结合，且一旦与正确的位点结合，即进行酶促地切割靶RNA。这类靶RNA的策略性切割将破坏其指导合成编码蛋白的能力。在酶促核酸结合和切割其RNA靶标之后，其从该RNA中释放以寻找另一个靶标且可反复结合和切割新靶标。In general, enzymatic nucleic acids with RNA cleavage activity work by first binding to a target RNA. Such binding occurs through the target binding portion of the enzymatic nucleic acid held in close proximity to the enzymatic portion of the molecule that cleaves the target RNA. Thus, the enzymatic nucleic acid first recognizes and then binds to the target RNA through complementary base pairing, and once bound to the correct site, enzymatically cleaves the target RNA. Strategic cleavage of such target RNAs would destroy their ability to direct the synthesis of encoded proteins. After an enzymatic nucleic acid binds and cleaves its RNA target, it is released from that RNA to seek another target and can repeatedly bind and cleave new targets.
已使用诸如体外选择(进化)策略等数种途径来进化能够催化各种反应的新核酸催化剂，所述反应为例如磷酸二酯键和酰胺键的切割和连接。Several approaches such as in vitro selection (evolution) strategies have been used to evolve new nucleic acid catalysts capable of catalyzing various reactions such as cleavage and ligation of phosphodiester and amide bonds.
催化活性最佳的核酶的开发会显著地有助于以调节基因表达为目的而采用RNA切割核酶的任何策略。例如锤头状核酶在存在Mg2+辅因子的饱和(10mM)浓度下，以约1min-1的催化速率(kcat)起作用。已显示人造“RNA连接酶”核酶以约100min-1的速率催化相应的自我修饰反应。此外，据了解具有由DNA组成的底物结合臂的某些经修饰的锤头状核酶，以接近100min-1的倍数转换速率(multiple turn-over rate)催化RNA切割。最终，用某些核苷酸类似物置换在锤头的催化核心内的特定残基产生显示出在催化速率上多达10倍改进的修饰核酶。这些研究结果证实核酶可以以显著高于大多数天然自我切割核酶展示于体外的催化速率，促进化学转化。那么可能的是，可优化某些自我切割核酶的结构以产生最高的催化活性，或者可制备展示出显著更快的RNA磷酸二酯切割速率的全新RNA基序。The development of ribozymes with optimal catalytic activity would significantly facilitate any strategy employing RNA-cleaving ribozymes for the purpose of modulating gene expression. For example, the hammerhead ribozyme acts with a catalytic rate (kcat) of about 1 min-1 in the presence of a saturating (10 mM) concentration of the Mg2+ cofactor. The artificial "RNA ligase" ribozyme has been shown to catalyze the corresponding self-modification reaction at a rate of about 100 min-1. Furthermore, certain modified hammerhead ribozymes with substrate-binding arms composed of DNA are known to catalyze RNA cleavage at multiple turn-over rates approaching 100 min-1. Ultimately, substitution of specific residues within the catalytic core of Hammerhead with certain nucleotide analogs resulted in modified ribozymes that exhibited as much as a 10-fold improvement in catalytic rate. These findings demonstrate that ribozymes can facilitate chemical transformations at catalytic rates significantly higher than those exhibited in vitro by most natural self-cleaving ribozymes. It is then possible that the structure of certain self-cleaving ribozymes could be optimized to yield the highest catalytic activity, or that entirely new RNA motifs could be made that exhibit significantly faster rates of RNA phosphodiester cleavage.
通过符合“锤头”模型的RNA催化剂来分子间切割RNA底物首先显示于1987年。将所述RNA催化剂回收且与多种RNA分子反应，证实其为真正催化性的。Intermolecular cleavage of RNA substrates by RNA catalysts fitting the "hammerhead" model was first shown in 1987. The RNA catalyst was recovered and reacted with a variety of RNA molecules, confirming that it was truly catalytic.
基于“锤头”基序设计的催化性RNA已通过在催化性RNA中作出适当的碱基改变以维持与靶序列的必要碱基配对，来用于切割特定靶序列。这允许使用催化性RNA来切割特定靶序列，并表明根据“锤头”模型设计的催化性RNA可能在体内切割特定底物RNA。Catalytic RNAs designed based on the "hammerhead" motif have been used to cleave specific target sequences by making appropriate base changes in the catalytic RNA to maintain the necessary base pairing with the target sequence. This allows the use of catalytic RNAs to cleave specific target sequences and suggests that catalytic RNAs designed according to the "hammerhead" model may cleave specific substrate RNAs in vivo.
RNA干扰(RNAi)已成为调节哺乳动物和哺乳动物细胞中基因表达的强大工具。此方法要求使用表达质粒或病毒以及加工成siRNA的小发夹RNA的编码序列，将小干扰RNA(siRNA)作为RNA本身或作为DNA递送。此系统能够有效将前siRNA转运到它们在其中活跃的细胞质中，并允许使用经调节的和组织特异性的启动子用于基因表达。RNA interference (RNAi) has emerged as a powerful tool for modulating gene expression in mammals and mammalian cells. This approach requires the delivery of small interfering RNA (siRNA) as RNA itself or as DNA, using expression plasmids or viruses and the coding sequence of small hairpin RNA processed into siRNA. This system enables efficient transport of pre-siRNAs into the cytoplasm where they are active and allows the use of regulated and tissue-specific promoters for gene expression.
在一个实施方案中，寡核苷酸或反义化合物包括核糖核酸(RNA)和/或脱氧核糖核酸(DNA)的寡聚体或多聚体、或其模拟物、嵌合体、类似物或同源物。此术语包括由天然存在核苷酸、糖和共价核苷间(骨架)键组成的寡核苷酸以及类似地起作用的具有非天然存在部分的寡核苷酸。由于所需性质，例如，增强的细胞摄取、对靶核酸增强的亲和力以及在核酸酶存在时增大的稳定性，常常需要这类经修饰或取代的寡核苷酸超过天然形式。In one embodiment, the oligonucleotide or antisense compound comprises an oligomer or polymer of ribonucleic acid (RNA) and/or deoxyribonucleic acid (DNA), or a mimetic, chimera, analog, or homolog thereof. Source material. The term includes oligonucleotides composed of naturally occurring nucleotides, sugars and covalent internucleoside (backbone) linkages as well as oligonucleotides with non-naturally occurring portions that function similarly. Such modified or substituted oligonucleotides are often desired over the native form due to desirable properties, eg, enhanced cellular uptake, enhanced affinity for target nucleic acids, and increased stability in the presence of nucleases.
根据本发明，寡核苷酸或“反义化合物”包括反义寡核苷酸(例如RNA、DNA、其模拟物、嵌合体、类似物或同源物)、核酶、外部指导序列(EGS)寡核苷酸、siRNA化合物、单链或双链RNA干扰(RNAi)化合物例如siRNA化合物、saRNA、aRNA，以及与靶核酸的至少一部分杂交且调节其功能的其它寡聚化合物。因此，它们可为DNA、RNA、DNA样、RNA样、或其混合物，或可为这些中的一种或更多种的模拟物。这些化合物可为单链、双链、环状或发夹寡聚化合物且可包含结构元件，例如内部或末端突起、错配或环。将反义化合物常规地制备成线性但可经连接或者另外地制备成环状和/或分枝的。反义化合物可包括构建体，例如杂交以形成完全或部分双链化合物的两条链，或带有足够自我互补性以允许杂交并形成完全或部分双链化合物的单链。可将所述两条链内部连接而留下游离的3’或5’末端或可将其连接形成连续的发夹结构或环。发夹结构可在5’或3’末端上包含突出端，产生单链特征的延伸。双链化合物任选可在末端上包含突出端。进一步的修饰可包括与末端之一、经挑选的核苷酸位置、糖位置或核苷间键之一连接的缀合基团。备选地，所述两条链可经由非核酸部分或连接基团来连接。当形成自仅一条链时，dsRNA可采取自我互补的发夹型分子形式，其在其自身上对折形成双链体。因此，所述dsRNA可为完全或部分双链的。基因表达的特异性调节可通过dsRNA发夹在转基因细胞系中的稳定表达来完成。当形成自两条链或采取在其自身上对折形成双链体的自身互补的发夹型分子形式的单链时，所述两条链(或单链的双链体形成区)为以沃森-克里克模式碱基配对的互补RNA链。According to the present invention, oligonucleotides or "antisense compounds" include antisense oligonucleotides (such as RNA, DNA, mimetics, chimeras, analogs or homologues thereof), ribozymes, external guide sequences (EGS ) oligonucleotides, siRNA compounds, single- or double-stranded RNA interference (RNAi) compounds such as siRNA compounds, saRNA, aRNA, and other oligomeric compounds that hybridize to at least a portion of a target nucleic acid and modulate its function. Thus, they may be DNA, RNA, DNA-like, RNA-like, or mixtures thereof, or may be mimetics of one or more of these. These compounds may be single-stranded, double-stranded, circular or hairpin oligomeric compounds and may contain structural elements such as internal or terminal protrusions, mismatches or loops. Antisense compounds are conventionally prepared linear but may be ligated or otherwise prepared circular and/or branched. Antisense compounds may include constructs such as two strands that hybridize to form a fully or partially double-stranded compound, or a single strand with sufficient self-complementarity to allow hybridization and form a fully or partially double-stranded compound. The two strands can be joined internally leaving free 3' or 5' ends or they can be joined to form a continuous hairpin structure or loop. The hairpin structure may contain an overhang on the 5' or 3' end, creating an extension that is characteristic of a single strand. Double-stranded compounds optionally may comprise overhangs on the ends. Further modifications may include a conjugation group attached to one of the termini, selected nucleotide positions, sugar positions or internucleoside linkages. Alternatively, the two strands may be linked via a non-nucleic acid moiety or linking group. When formed from only one strand, dsRNA can take the form of a self-complementary hairpin-type molecule that folds on itself to form a duplex. Thus, the dsRNA can be fully or partially double-stranded. Specific regulation of gene expression can be accomplished by stable expression of dsRNA hairpins in transgenic cell lines. When formed from two strands or a single strand in the form of a self-complementary hairpin molecule that folds on itself to form a duplex, the two strands (or the duplex-forming region of the single strand) are separated by Complementary RNA strands that are base-paired in the Sen-Crick pattern.
一旦引入系统，本发明的化合物可引起一种或更多种酶或结构蛋白的作用以实现靶核酸的切割或其它修饰，或可经由基于占据的机制来运作。一般而言，核酸(包括寡核苷酸)可描述为“DNA样”(即，一般具有一个或更多个2’脱氧糖和，一般地，T而不是U碱基)或“RNA样”(即，一般具有一个或更多个2’羟基或2’修饰的糖和，一般U而不是T碱基)。核酸螺旋可采取多于一种类型的结构，最普通地A和B型。据认为，一般而言，具有B型样结构的寡核苷酸为“DNA样”而具有A型样结构的寡核苷酸为“RNA样”。在一些(嵌合的)实施方案中，反义化合物可包含A和B型区两者。Once introduced into the system, the compounds of the invention may cause the action of one or more enzymes or structural proteins to effect cleavage or other modification of the target nucleic acid, or may operate via an occupancy-based mechanism. In general, nucleic acids (including oligonucleotides) can be described as "DNA-like" (i.e., generally having one or more 2' deoxysugars and, generally, T rather than U bases) or "RNA-like" (ie, generally have one or more 2' hydroxyl or 2' modified sugars and, generally U rather than T bases). Nucleic acid helices can adopt more than one type of structure, most commonly A and B types. In general, oligonucleotides with a B-like structure are considered "DNA-like" and oligonucleotides with an A-like structure are "RNA-like". In some (chimeric) embodiments, an antisense compound may comprise both A and B-type regions.
根据本发明的反义化合物可包含约5-约80个核苷酸(即约5-约80个连接核苷)长度的反义部分。这是指反义化合物的反义链或部分的长度。换言之，本发明的单链反义化合物包含5-约80个核苷酸，而本发明的双链反义化合物(例如，dsRNA)包含5-约80个核苷酸长度的有义和反义链或部分。本领域普通技术人员将认识到，这包括5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79或80个核苷酸长度、或其之内任何范围的反义部分。Antisense compounds according to the invention may comprise an antisense portion of about 5 to about 80 nucleotides (ie, about 5 to about 80 linked nucleosides) in length. This refers to the length of the antisense strand or portion of the antisense compound. In other words, the single-stranded antisense compounds of the invention comprise 5 to about 80 nucleotides, while the double-stranded antisense compounds (e.g., dsRNA) of the invention comprise sense and antisense sequences of 5 to about 80 nucleotides in length. chain or part. Those of ordinary skill in the art will recognize that this includes 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 , 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 , 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74 , 75, 76, 77, 78, 79 or 80 nucleotides in length, or any range within the antisense portion.
在一个实施方案中，本发明的反义化合物具有10-50个核苷酸长度的反义部分。本领域普通技术人员将认识到，这包括具有10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49或50个核苷酸长度、或其之内任何范围的反义部分的寡核苷酸。在一些实施方案中，寡核苷酸长度为15个核苷酸。In one embodiment, the antisense compounds of the invention have an antisense portion of 10-50 nucleotides in length. Those of ordinary skill in the art will recognize that this includes having 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length, or An oligonucleotide of any range within the antisense portion. In some embodiments, the oligonucleotide is 15 nucleotides in length.
在一个实施方案中，本发明的反义或寡核苷酸化合物具有12或13-30个核苷酸长度的反义部分。本领域普通技术人员将认识到，这包括具有12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29或30个核苷酸长度、或其之内任何范围的反义部分的反义化合物。In one embodiment, the antisense or oligonucleotide compounds of the invention have an antisense portion that is 12 or 13-30 nucleotides in length. Those of ordinary skill in the art will recognize that this includes having 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 Antisense compounds of antisense moieties within nucleotide lengths, or any range thereof.
在另一个实施方案中，本发明的寡聚化合物也包括其中不同的碱基存在于化合物中的一个或更多个核苷酸位置上的变体。例如，如果第一个核苷酸为腺苷，那么可产生在此位置包含胸苷、鸟苷或胞苷的变体。这可在反义或dsRNA化合物的任何位置上进行。然后使用本文所述的方法来检测这些化合物以确定其抑制靶核酸表达的能力。In another embodiment, the oligomeric compounds of the invention also include variants wherein a different base is present at one or more nucleotide positions in the compound. For example, if the first nucleotide is adenosine, variants can be produced that contain thymidine, guanosine, or cytidine at this position. This can be done at any position on the antisense or dsRNA compound. These compounds are then tested using the methods described herein to determine their ability to inhibit expression of the target nucleic acid.
在有些实施方案中，反义化合物与靶标之间的同源性、序列同一性或互补性是约40％至约60％。在有些实施方案中，同源性、序列同一性或互补性是约60％至约70％。在有些实施方案中，同源性、序列同一性或互补性是约70％至约80％。在有些实施方案中，同源性、序列同一性或互补性是约80％至约90％。在有些实施方案中，同源性、序列同一性或互补性是约90％、约92％、约94％、约95％、约96％、约97％、约98％、约99％或约100％。In some embodiments, the homology, sequence identity or complementarity between the antisense compound and the target is about 40% to about 60%. In some embodiments, the homology, sequence identity or complementarity is about 60% to about 70%. In some embodiments, the homology, sequence identity or complementarity is about 70% to about 80%. In some embodiments, the homology, sequence identity or complementarity is about 80% to about 90%. In some embodiments, the homology, sequence identity or complementarity is about 90%, about 92%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%.
在另一个实施方案中，反义寡核苷酸(例如，SEQ ID NO：4-30中所述的核酸分子)包含一个或更多个取代或修饰。在一个实施方案中，将核苷酸用锁定核酸(LNA)取代。In another embodiment, an antisense oligonucleotide (eg, a nucleic acid molecule set forth in SEQ ID NO: 4-30) comprises one or more substitutions or modifications. In one embodiment, the nucleotides are replaced with locked nucleic acids (LNA).
在另一个实施方案中，寡核苷酸靶向与抗病毒基因有关的编码和/或非编码序列以及如SEQ ID NO：1-9所述的序列的有义和/或反义的核酸分子的一个或更多个区。也将寡核苷酸靶向SEQ ID NO：1-9的重叠区。In another embodiment, the oligonucleotides target the coding and/or non-coding sequences associated with antiviral genes and the sense and/or antisense nucleic acid molecules of the sequences set forth in SEQ ID NO: 1-9 one or more districts. Oligonucleotides were also targeted to the overlapping regions of SEQ ID NO: 1-9.
本发明的某些寡核苷酸为嵌合寡核苷酸。“嵌合寡核苷酸”或“嵌合体”，在本发明的背景中，为包含两个或更多个化学上不同区的寡核苷酸，每个区由至少一个核苷酸组成。这些寡核苷酸典型地包含赋予一种或更多种有益特性(例如，对核酸酶的抗性增强、摄入细胞增强、对靶标增强的结合亲和力)的经修饰的核苷酸的至少一个区，以及作为能够切割RNA:DNA或RNA:RNA杂合体的酶底物的区。作为实例，核糖核酸酶H为细胞核酸内切酶，其切割RNA:DNA双链体的RNA链。核糖核酸酶H的活化因此导致RNA靶标的切割，从而大大地增强基因表达的反义调节效率。因此，当使用嵌合寡核苷酸时，与杂交到相同靶区的硫代磷酸酯脱氧寡核苷酸相比，常常可用较短的寡核苷酸获得相当的结果。RNA靶标的切割可通过凝胶电泳和必要时本领域已知的相关核酸杂交技术，常规地检测。在一个实施方案中，嵌合寡核苷酸包含修饰成增加靶结合亲和力的至少一个区，并且通常包含充当核糖核酸酶H的底物的区。寡核苷酸对其靶标(在此情况下，编码ras的核酸)的亲和力通过测定寡核苷酸/靶标对的Tm来常规地确定，Tm为寡核苷酸与靶标解离的温度；解离以分光光度法检测。Tm越高，寡核苷酸对靶标的亲和力越大。Certain oligonucleotides of the invention are chimeric oligonucleotides. A "chimeric oligonucleotide" or "chimera", in the context of the present invention, is an oligonucleotide comprising two or more chemically distinct regions, each region consisting of at least one nucleotide. These oligonucleotides typically comprise at least one of the modified nucleotides that confer one or more beneficial properties (e.g., increased resistance to nucleases, enhanced cellular uptake, enhanced binding affinity for the target) regions, and regions that are substrates for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. As an example, RNase H is a cellular endonuclease that cleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H thus results in cleavage of the RNA target, thereby greatly enhancing the efficiency of antisense regulation of gene expression. Thus, comparable results can often be obtained with shorter oligonucleotides when chimeric oligonucleotides are used compared to phosphorothioate deoxyoligonucleotides that hybridize to the same target region. Cleavage of RNA targets can be routinely detected by gel electrophoresis and, if necessary, related nucleic acid hybridization techniques known in the art. In one embodiment, the chimeric oligonucleotide comprises at least one region modified to increase target binding affinity, and typically comprises a region that acts as a substrate for RNase H. The affinity of an oligonucleotide for its target (in this case, a ras-encoding nucleic acid) is routinely determined by determining the Tm of the oligonucleotide/target pair, which is the temperature at which the oligonucleotide dissociates from the target; detected by spectrophotometry. The higher the Tm, the greater the affinity of the oligonucleotide for the target.
本发明的嵌合反义化合物可作为如上所述的两个或更多个寡核苷酸、修饰的寡核苷酸、寡聚核苷和/或寡核苷酸模拟物的复合结构而形成。本领域亦已将这类化合物称为杂合体或gapmer。教导制备这类杂合结构的代表性美国专利包括但不限于，美国专利第5,013,830、5,149,797、5,220,007、5,256,775、5,366,878、5,403,711、5,491,133、5,565,350、5,623,065、5,652,355、5,652,356和5,700,922号，它们各自通过引用并入本文。Chimeric antisense compounds of the invention can be formed as composite structures of two or more oligonucleotides, modified oligonucleotides, oligonucleosides and/or oligonucleotide mimetics as described above . Such compounds have also been referred to in the art as hybrids or gapmers. Representative U.S. patents that teach the preparation of such hybrid structures include, but are not limited to, U.S. Patent Nos. 5,013,830, 5,149,797, 5,220,007, 5,256,775, 5,366,878, 5,403,711, 5,491,133, 5,565,350, 5,623,065, 5,652,355, 5,652,30, each of which is incorporated by reference in 2,652,35 into this article.
在另一个实施方案中，经修饰的寡核苷酸区包含在糖的2’位置上修饰的至少一个核苷酸，最优选2’-O烷基、2’-O-烷基-O-烷基或2’-氟修饰的核苷酸。在其它实施方案中，RNA修饰包括在RNA 3’末端的嘧啶、脱碱基残基或反向碱基的核糖上的2’-氟、2’-氨基和2’O-甲基修饰。将这类修饰常规地掺入到寡核苷酸中，已显示这些寡核苷酸具有比2’-脱氧寡核苷酸对给定靶标更高的Tm(即，更高的靶结合亲和力)。这种增加的亲和力的作用为大大地增强基因表达的RNAi寡核苷酸抑制。核糖核酸酶H为切割RNA:DNA双链体的RNA链的细胞核酸内切酶；此酶的活化因此导致RNA靶标的切割，且因此可大大地增强RNAi抑制效率。RNA靶标的切割可通过凝胶电泳来常规地证实。在另一个实施方案中，也修饰嵌合寡核苷酸以增强核酸酶抗性。细胞包含可降解核酸的各种核酸外切酶和核酸内切酶。已显示许多核苷酸和核苷修饰使掺入有它们的寡核苷酸比天然寡脱氧核苷酸对核酸酶消化更有抗性。核酸酶抗性通过将寡核苷酸与细胞提取物或分离的核酸酶溶液一起温育并测定随时间推移剩余的完好寡核苷酸的程度(通常地通过凝胶电泳)来常规地测定。已修饰成增强其核酸酶抗性的寡核苷酸比未修饰的寡核苷酸保持完好持续更长时间。已证实多种寡核苷酸修饰增强或赋予核酸酶抗性。包含至少一个硫代磷酸酯修饰的寡核苷酸为目前更优选的。在一些情况下，增强靶结合亲和力的寡核苷酸修饰也能够独立地增强核酸酶抗性。一些所需的修饰可在De Mesmaeker等，(1995)Acc.Chem.Res.，28：366-374中找到。In another embodiment, the modified oligonucleotide region comprises at least one nucleotide modified at the 2' position of the sugar, most preferably 2'-Oalkyl, 2'-O-alkyl-O- Alkyl or 2'-fluoro modified nucleotides. In other embodiments, RNA modifications include 2'-fluoro, 2'-amino, and 2'O-methyl modifications on pyrimidines, abasic residues, or the ribose sugar of the inverted base at the 3' end of the RNA. Such modifications are routinely incorporated into oligonucleotides that have been shown to have a higher Tm (i.e., higher target binding affinity) for a given target than 2'-deoxyoligonucleotides . The effect of this increased affinity is to greatly enhance RNAi oligonucleotide inhibition of gene expression. RNase H is a cellular endonuclease that cleaves the RNA strand of an RNA:DNA duplex; activation of this enzyme thus results in cleavage of the RNA target and thus can greatly enhance RNAi inhibition efficiency. Cleavage of RNA targets can be routinely confirmed by gel electrophoresis. In another embodiment, the chimeric oligonucleotides are also modified to enhance nuclease resistance. Cells contain various exonucleases and endonucleases that degrade nucleic acids. A number of nucleotide and nucleoside modifications have been shown to make oligonucleotides into which they are incorporated more resistant to nuclease digestion than natural oligodeoxynucleotides. Nuclease resistance is routinely determined by incubating oligonucleotides with cell extracts or isolated nuclease solutions and determining the extent of intact oligonucleotides remaining over time, typically by gel electrophoresis. Oligonucleotides that have been modified to enhance their nuclease resistance remain intact for longer periods of time than unmodified oligonucleotides. A variety of oligonucleotide modifications have been shown to enhance or confer nuclease resistance. Oligonucleotides comprising at least one phosphorothioate modification are presently more preferred. In some cases, oligonucleotide modifications that enhance target binding affinity can also independently enhance nuclease resistance. Some desirable modifications can be found in De Mesmaeker et al., (1995) Acc. Chem. Res., 28:366-374.
预想用于本发明的一些寡核苷酸的具体实例包括包含经修饰的骨架的那些，所述经修饰的骨架为例如硫代磷酸酯、磷酸三酯、甲基膦酸酯、短链烷基或环烷基糖间键或者短链杂原子或杂环的糖间键。最优选的为带有硫代磷酸酯骨架和带有杂原子骨架的寡核苷酸，特别地CH2--NH--O--CH2、CH，--N(CH3)--O--CH2[称为亚甲基(甲亚氨基)或MMI骨架]、CH2--O--N(CH3)--CH2、CH2-N(CH3)--N(CH3)--CH2和O--N(CH3)--CH2--CH2骨架，其中天然磷酸二酯骨架表示为O--P--O-CH。由De Mesmaeker等，(1995)Acc.Chem.Res.28：366-374公开的酰胺骨架也为优选的。同样优选的为具有吗啉代骨架结构的寡核苷酸(Summerton和Weller，美国专利第5,034,506号)。在其它实施方案中，将寡核苷酸的例如肽核酸(PNA)骨架、磷酸二酯骨架替换为聚酰胺骨架，核苷酸直接或间接地与聚酰胺骨架的氮杂氮原子结合。寡核苷酸也可包含一个或更多个取代的糖部分。寡核苷酸在2’位置上包含下列中的一种：OH、SH、SCH3、F、OCN、OCH3OCH3、OCH3O(CH2)n CH3、O(CH2)n NH2或O(CH2)n CH3，其中n为1-约10；C1-C10低级烷基、烷氧基烷氧基、取代的低级烷基、烷芳基或芳烷基；Cl；Br；CN；CF3；OCF3；O--、S--、或N-烷基；O--、S--、或N-烯基；SOCH3；SO2CH3；ONO2；NO2；N3；NH2；杂环烷基；杂环烷芳基；氨基烷基氨基；聚烷基氨基；取代的甲硅烷基；RNA切割基团；报道基团；嵌入剂；改进寡核苷酸药代动力学特性的基团；或改进寡核苷酸药效学特性的基团以及具有类似特性的其它取代基。修饰包括2’-甲氧乙氧基[2′-O-CH2 CH2 OCH3，也称为2′-O-(2-甲氧乙基)]。其它修饰包括2’-甲氧基(2′-O--CH3)、2’-丙氧基(2′-OCH2CH2CH3)和2’-氟(2′-F)。类似的修饰也可在寡核苷酸的其它位置上进行，具体地在3’末端核苷酸上糖的3’位置和5’末端核苷酸的5’位置。寡核苷酸也可具有糖模拟物例如取代戊呋喃糖基基团的环丁基。Specific examples of some oligonucleotides envisioned for use in the present invention include those comprising modified backbones such as phosphorothioate, phosphotriester, methylphosphonate, short chain alkyl Or cycloalkyl sugar linkages or short-chain heteroatoms or heterocyclic sugar linkages. Most preferred are oligonucleotides with a phosphorothioate backbone and with a heteroatom backbone, especially CH2--NH--O--CH2, CH,--N(CH3)--O--CH2 [known as methylene (formimino) or MMI skeleton], CH2--O--N(CH3)--CH2, CH2-N(CH3)--N(CH3)--CH2 and O--N (CH3)--CH2--CH2 skeleton, wherein the natural phosphodiester skeleton is expressed as O--P--O-CH. The amide backbone disclosed by De Mesmaeker et al., (1995) Acc. Chem. Res. 28:366-374 is also preferred. Also preferred are oligonucleotides having a morpholino backbone structure (Summerton and Weller, US Patent No. 5,034,506). In other embodiments, the eg peptide nucleic acid (PNA) backbone, phosphodiester backbone of the oligonucleotide is replaced with a polyamide backbone, and the nucleotides are directly or indirectly bonded to the aza nitrogen atoms of the polyamide backbone. An oligonucleotide may also contain one or more substituted sugar moieties. The oligonucleotide contains one of the following at the 2' position: OH, SH, SCH3, F, OCN, OCH3OCH3, OCH3O(CH2)n CH3, O(CH2)n NH2 or O(CH2)n CH3, where n is 1 to about 10; C1-C10 lower alkyl, alkoxyalkoxy, substituted lower alkyl, alkaryl or aralkyl; Cl; Br; CN; CF3; OCF3; --, or N-alkyl; O--, S--, or N-alkenyl; SOCH3; SO2CH3; ONO2; NO2; N3; NH2; heterocycloalkyl; heterocycloalkaryl; aminoalkylamino ; polyalkylamino; substituted silyl groups; RNA cleavage groups; reporter groups; intercalators; groups that improve oligonucleotide pharmacokinetic properties; or groups that improve oligonucleotide pharmacodynamic properties groups and other substituents with similar properties. Modifications include 2'-methoxyethoxy [2'-O-CH2 CH2 OCH3, also known as 2'-O-(2-methoxyethyl)]. Other modifications include 2'-methoxy (2'-O--CH3), 2'-propoxy (2'-OCH2CH2CH3) and 2'-fluoro (2'-F). Similar modifications can also be made at other positions of the oligonucleotide, specifically at the 3' position of the sugar on the 3' terminal nucleotide and the 5' position of the 5' terminal nucleotide. Oligonucleotides may also have sugar mimetics such as cyclobutyl in place of the pentofuranosyl group.
寡核苷酸也可另外或备选地包含核碱基(本领域常常简称为“碱基”)修饰或取代。本文所用的“未修饰的”或“天然的”核苷酸包括腺嘌呤(A)、鸟嘌呤(G)、胸腺嘧啶(T)、胞嘧啶(C)和尿嘧啶(U)。经修饰的核苷酸包括在天然核酸中仅稀少或短暂地存在的核苷酸，例如，次黄嘌呤、6-甲基腺嘌呤、5-Me嘧啶、特别地5-甲基胞嘧啶(也称为5-甲基-2’脱氧胞嘧啶且常常在本领域中称为5-Me-C)、5-羟甲基胞嘧啶(HMC)、糖基HMC和龙胆二糖基HMC，以及合成核苷酸，例如，2-氨基腺嘌呤、2-(甲氨基)腺嘌呤、2-(咪唑基烷基)腺嘌呤、2-(氨烷基氨基)腺嘌呤或其它杂取代的烷基腺嘌呤、2-硫尿嘧啶、2-硫胸腺嘧啶、5-溴尿嘧啶、5-羟基甲基尿嘧啶、8-氮杂鸟嘌呤、7-脱氮鸟嘌呤、N6(6-氨己基)腺嘌呤和2，6-二氨基嘌呤。可包括本领域已知的“通用的”碱基，例如，肌苷。已显示5-Me-C取代增强核酸双链体的稳定性达0.6-1.2℃(Sanghvi，Y.S.，载于Crooke，S.T.和Lebleu，B.，编辑，Antisense Research andApplications，CRC Press，Boca Raton，1993，276-278页)，且为目前的碱基取代。Oligonucleotides may also additionally or alternatively comprise nucleobase (often referred to in the art simply as "base") modifications or substitutions. As used herein, "unmodified" or "natural" nucleotides include adenine (A), guanine (G), thymine (T), cytosine (C) and uracil (U). Modified nucleotides include nucleotides that occur only rarely or transiently in natural nucleic acids, for example, hypoxanthine, 6-methyladenine, 5-Me pyrimidine, especially 5-methylcytosine (also known as 5-methyl-2'deoxycytosine and often referred to in the art as 5-Me-C), 5-hydroxymethylcytosine (HMC), glycosyl HMC, and gentiobiosyl HMC, and Synthetic nucleotides, e.g., 2-aminoadenine, 2-(methylamino)adenine, 2-(imidazolylalkyl)adenine, 2-(aminoalkylamino)adenine, or other heterosubstituted alkyl Adenine, 2-thiouracil, 2-thiothymine, 5-bromouracil, 5-hydroxymethyluracil, 8-azaguanine, 7-deazaguanine, N6(6-aminohexyl) Adenine and 2,6-diaminopurine. "Universal" bases known in the art may be included, eg, inosine. The 5-Me-C substitution has been shown to enhance the stability of nucleic acid duplexes up to 0.6-1.2°C (Sanghvi, Y.S., in Crooke, S.T. and Lebleu, B., eds., Antisense Research and Applications, CRC Press, Boca Raton, 1993 , pp. 276-278), and is the current base substitution.
本发明的寡核苷酸的另一种修饰涉及以化学方法使一种或更多种增强寡核苷酸的活性或细胞摄取的部分或缀合物与寡核苷酸连接。这类部分包括但不限于：脂质部分，例如胆固醇部分、胆甾醇基部分、硫醚例如己基-S-三苯甲基硫醇、硫代胆固醇、脂族链例如十二烷二醇或十一烷基残基、磷脂例如二-十六烷基-外消旋-甘油或1，2-二-O-十六烷基-外消旋-甘油-3-H-膦酸三乙铵、聚胺或聚乙二醇链、或金刚烷乙酸。包含亲脂性部分的寡核苷酸以及用于制备这类寡核苷酸的方法为本领域已知的，例如美国专利第5,138,045、5,218,105和5,459,255号。Another modification of the oligonucleotides of the invention involves chemically attaching to the oligonucleotide one or more moieties or conjugates that enhance the activity or cellular uptake of the oligonucleotide. Such moieties include, but are not limited to: lipid moieties such as cholesterol moieties, cholesteryl moieties, thioethers such as hexyl-S-tritylthiol, thiocholesterol, aliphatic chains such as dodecanediol or decane Monoalkyl residues, phospholipids such as di-hexadecyl-rac-glycerol or 1,2-di-O-hexadecyl-rac-glycerol-3-H-phosphonic acid triethylammonium, Polyamine or polyethylene glycol chains, or adamantane acetic acid. Oligonucleotides comprising lipophilic moieties and methods for preparing such oligonucleotides are known in the art, eg, US Patent Nos. 5,138,045, 5,218,105, and 5,459,255.
无需将给定寡核苷酸中的所有位置一致地修饰，且实际上多于一种上述修饰可掺入到单个寡核苷酸中或甚至在寡核苷酸内的单个核苷内部。本发明也包括作为如上文中定义的嵌合寡核苷酸的寡核苷酸。All positions in a given oligonucleotide need not be uniformly modified, and in fact more than one such modification may be incorporated into a single oligonucleotide or even within a single nucleoside within an oligonucleotide. The invention also includes oligonucleotides which are chimeric oligonucleotides as defined above.
在另一个实施方案中，本发明的核酸分子与另一个部分缀合，所述部分包括但不限于脱碱基核苷酸、聚醚、聚胺、聚酰胺、肽、碳水化合物、脂质或聚碳氢化合物。本领域技术人员将认识到，可将这些分子在糖、碱基或磷酸基的数个位置上连接到一个或更多个构成核酸分子的任何核苷酸。In another embodiment, a nucleic acid molecule of the invention is conjugated to another moiety including, but not limited to, abasic nucleotides, polyethers, polyamines, polyamides, peptides, carbohydrates, lipids or polyhydrocarbons. Those skilled in the art will recognize that these molecules can be linked to one or more of any of the nucleotides making up the nucleic acid molecule at several positions on the sugar, base or phosphate group.
根据本发明使用的寡核苷酸可通过众所周知的固相合成技术来便利和常规地制备。用于这类合成的设备由包括Applied Biosystems在内的数个供应商销售。也可使用用于这类合成的任何其它方法；寡核苷酸的实际合成完全在本领域普通技术人员的才能之内。亦众所周知的是使用类似技术来制备其它寡核苷酸，例如硫代磷酸酯和烷基化衍生物。还众所周知的是使用类似技术和市购经修饰的酰胺化物(amidite)和可控孔度玻璃(CPG)产品，例如生物素、荧光黄、吖啶、或补骨脂素修饰的酰胺化物和/或CPG(可从GlenResearch，Sterling VA购买)，以合成荧光标记的、生物素化的或其它修饰的寡核苷酸，例如胆固醇修饰的寡核苷酸。Oligonucleotides for use in accordance with the present invention can be conveniently and routinely prepared by well known techniques of solid phase synthesis. Equipment for such syntheses is sold by several suppliers including Applied Biosystems. Any other method for such synthesis may also be used; the actual synthesis of oligonucleotides is well within the purview of one of ordinary skill in the art. It is also well known to use similar techniques to prepare other oligonucleotides, such as phosphorothioate and alkylated derivatives. It is also well known to use similar techniques and commercially available modified amidite and controlled pore glass (CPG) products such as biotin, fluorescein, acridine, or psoralen modified amidite and/or or CPG (available from GlenResearch, Sterling VA) to synthesize fluorescently labeled, biotinylated, or otherwise modified oligonucleotides, such as cholesterol-modified oligonucleotides.
根据本发明，使用修饰(例如使用LNA单体)以增加寡核苷酸的效价、特异性和作用持续时间并拓宽其施用途径，所述寡核苷酸包含诸如MOE、ANA、FANA、PS等当前的化学物质。这可通过用LNA单体取代当前寡核苷酸中的一些单体来完成。LNA修饰的寡核苷酸可具有类似于母体化合物的大小或可更大或优选更小。这类LNA修饰的寡核苷酸包含少于约70％、更优选少于约60％、最优选少于约50％的LNA单体且其大小在约5-25个核苷酸之间，更优选在约12-20个核苷酸之间。According to the invention, modifications are used (for example using LNA monomers) to increase the potency, specificity and duration of action of oligonucleotides comprising such as MOE, ANA, FANA, PS and to broaden their route of administration. and other current chemicals. This can be accomplished by substituting LNA monomers for some of the monomers in the current oligonucleotide. LNA modified oligonucleotides may be of similar size to the parent compound or may be larger or preferably smaller. Such LNA-modified oligonucleotides comprise less than about 70%, more preferably less than about 60%, most preferably less than about 50% LNA monomers and are between about 5-25 nucleotides in size, More preferably between about 12-20 nucleotides.
修饰的寡核苷酸骨架包括但不限于硫代磷酸酯、手性硫代磷酸酯、二硫代磷酸酯、磷酸三酯、氨烷基磷酸三酯、甲基和其它烷基膦酸酯，包括3’烯基膦酸酯和手性膦酸酯、次膦酸酯、氨基磷酸酯，包括3’-氨基氨基磷酸酯和氨烷基氨基磷酸酯、硫羰基氨基磷酸酯、硫羰基烷基膦酸酯、硫羰基烷基磷酸三酯，以及具有正常3’-5’键合的硼烷磷酸酯(boranophosphates)、这些的2’-5’连接类似物，以及具有反极性的那些，其中核苷单元的相邻对为3’-5’与5’-3’或2’-5’与5’-2’连接。也包括各种盐、混合盐和游离酸形式。Modified oligonucleotide backbones include, but are not limited to, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkylphosphonates, Including 3' alkenyl phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3'-aminophosphoramidates and aminoalkyl phosphoramidates, thiocarbonyl phosphoramidates, thiocarbonyl alkyl Phosphonates, thiocarbonylalkyl phosphate triesters, and boranophosphates with normal 3'-5' linkage, 2'-5' linked analogs of these, and those with reversed polarity, Wherein the adjacent pairs of nucleoside units are connected by 3'-5' and 5'-3' or 2'-5' and 5'-2'. Also included are various salts, mixed salts and free acid forms.
教导制备上述含磷键的代表性的美国专利包括但不限于，美国专利第3,687,808、4,469,863、4,476,301、5,023,243、5,177,196、5,188,897、5,264,423、5,276,019、5,278,302、5,286,717、5,321,131、5,399,676、5,405,939、5,453,496、5,455,233、5,466,677、5,476,925、5,519,126、5,536,821、5,541,306、5,550,111、5,563,253、5,571,799、5,587,361和5,625,050号，它们各自通过引用并入本文。教导制备上述含磷键的代表性的美国专利包括但不限于，美国专利第3,687,808、4,469,863、4,476,301、5,023,243、5,177,196、5,188,897、5,264,423、5,276,019、5,278,302、5,286,717、5,321,131、5,399,676、5,405,939、5,453,496、5,455,233、 5,466,677, 5,476,925, 5,519,126, 5,536,821, 5,541,306, 5,550,111, 5,563,253, 5,571,799, 5,587,361, and 5,625,050, each of which is incorporated herein by reference.
修饰的寡核苷酸骨架(其中不包含磷原子)，具有由短链烷基或环烷基核苷间键、混合杂原子和烷基或环烷基核苷间键、或一种或更多种短链杂原子或杂环核苷间键形成的骨架。这些包括具有吗啉代键的骨架(部分由核苷的糖部分形成)；硅氧烷骨架；硫化物、亚砜和砜骨架；甲乙酰基(formacetyl)和硫代甲乙酰基(thioformacetyl)骨架；亚甲基甲乙酰基和硫代甲乙酰基骨架；含烯骨架；氨基磺酸酯骨架；亚甲基亚氨基和亚甲基肼基骨架；磺酸酯或氨磺酰骨架；酰胺骨架；以及具有混合N、O、S和CH2组分部分的其它骨架。Modified oligonucleotide backbones (which do not contain phosphorus atoms) having short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatoms and alkyl or cycloalkyl internucleoside linkages, or one or more A backbone formed by bonds between various short-chain heteroatoms or heterocyclic nucleosides. These include backbones with morpholino linkages (formed in part from the sugar moieties of nucleosides); siloxane backbones; sulfide, sulfoxide, and sulfone backbones; formacetyl and thioformacetyl backbones; Methylformyl and thioformyl skeletons; alkene-containing skeletons; sulfamate skeletons; methyleneimino and methylenehydrazine skeletons; sulfonate or sulfonamide skeletons; , O, S and other skeletons of the CH2 component part.
教导制备上述寡聚核苷的代表性的美国专利包括但不限于，美国专利第5,034,506、5,166,315、5,185,444、5,214,134、5,216,141、5,235,033、5,264,562、5,264,564、5,405,938、5,434,257、5,466,677、5,470,967、5,489,677、5,541,307、5,561,225、5,596,086、5,602,240、5,610,289、5,602,240、5,608,046、5,610,289、5,618,704、5,623,070、5,663,312、5,633,360、5,677,437和5,677,439号，它们各自通过引用并入本文。教导制备上述寡聚核苷的代表性的美国专利包括但不限于，美国专利第5,034,506、5,166,315、5,185,444、5,214,134、5,216,141、5,235,033、5,264,562、5,264,564、5,405,938、5,434,257、5,466,677、5,470,967、5,489,677、5,541,307、5,561,225 , 5,596,086, 5,602,240, 5,610,289, 5,602,240, 5,608,046, 5,610,289, 5,618,704, 5,623,070, 5,663,312, 5,633,360, 5,677,437, and 5,677,439, each of which is incorporated herein by reference.
在其它寡核苷酸模拟物中，将核苷酸单元的糖和核苷间键(即骨架)，均用新基团置换。维持碱基单元用于与适当的核酸靶化合物杂交。一种这类寡聚化合物(已显示具有优秀的杂交特性的寡核苷酸模拟物)，称为肽核酸(PNA)。在PNA化合物中，寡核苷酸的糖骨架替换为含酰胺的骨架，具体地氨乙基甘氨酸骨架。将所述核碱基保留并直接或间接地与骨架的酰胺部分的氮杂氮原子结合。教导制备PNA化合物的代表性的美国专利包括但不限于，美国专利第5,539,082、5,714,331和5,719,262号，它们各自通过引用并入本文。PNA化合物的进一步教导可在Nielsen等，(1991)Science 254，1497-1500中找到。In other oligonucleotide mimetics, both the sugar and the internucleoside linkage (ie, the backbone) of the nucleotide unit are replaced with new groups. The base unit is maintained for hybridization to the appropriate nucleic acid target compound. One such oligomeric compound, an oligonucleotide mimic that has been shown to have excellent hybridization properties, is known as peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of the oligonucleotide is replaced with an amide-containing backbone, specifically an aminoethylglycine backbone. The nucleobase is retained and bonded directly or indirectly to the aza nitrogen atom of the amide portion of the backbone. Representative US patents that teach the preparation of PNA compounds include, but are not limited to, US Patent Nos. 5,539,082, 5,714,331, and 5,719,262, each of which is incorporated herein by reference. Further teaching on PNA compounds can be found in Nielsen et al., (1991) Science 254, 1497-1500.
在本发明的另一个实施方案中，带有硫代磷酸酯骨架的寡核苷酸和带有杂原子骨架的寡聚核苷，具体地-CH2-NH-O-CH2-，-CH2-N(CH3)-O-CH2-(称为亚甲基(甲亚氨基)或MMI骨架)，-CH2-O-N(CH3)-CH2-、-CH2N(CH3)-N(CH3)CH2-和-O-N(CH3)-CH2-CH2-，其中天然磷酸二酯骨架表示为上文引用的美国专利第5,489,677号的-O-P-O-CH2-，以及上文引用的美国专利第5,602,240号的酰胺骨架。还有具有上文引用的美国专利第5,034,506号的吗啉代骨架结构的寡核苷酸。In another embodiment of the present invention, oligonucleotides with a phosphorothioate backbone and oligonucleosides with a heteroatom backbone, specifically -CH2-NH-O-CH2-, -CH2-N (CH3)-O-CH2- (known as methylene (methylimino) or MMI skeleton), -CH2-O-N(CH3)-CH2-, -CH2N(CH3)-N(CH3)CH2- and -O-N (CH3)-CH2-CH2-, wherein the natural phosphodiester backbone is represented as -O-P-O-CH2- of US Patent No. 5,489,677 cited above, and the amide backbone of US Patent No. 5,602,240 cited above. There are also oligonucleotides having the morpholino backbone structure of US Patent No. 5,034,506 cited above.
修饰的寡核苷酸也可包含一个或更多个经取代的糖部分。寡核苷酸在2’位置上包含下列中的一种：OH；F；O-、S-、或N-烷基；O-、S-、或N-烯基；O-、S-或N-炔基；或O烷基-O-烷基，其中所述烷基、烯基和炔基可为取代或未取代的C到CO烷基或C2到CO烯基和炔基。特别是O(CH2)n OmCH3、O(CH2)n、OCH3、O(CH2)nNH2、O(CH2)nCH3、O(CH2)nONH2和O(CH2nON(CH2)nCH3)2，其中n和m可为1-约10。其它寡核苷酸在2’位置上包含下列中的一种：C到CO、低级烷基、取代的低级烷基、烷芳基、芳烷基、O-烷芳基或O-芳烷基、SH、SCH3、OCN、Cl、Br、CN、CF3、OCF3、SOCH3、SO2CH3、ONO2、NO2、N3、NH2、杂环烷基、杂环烷芳基、氨烷基氨基、聚烷基氨基、取代的甲硅烷基、RNA切割基团、报道基团、嵌入剂、用于改进寡核苷酸药代动力学特性的基团、或用于改进寡核苷酸药效学特性的基团，以及具有类似特性的其它取代基。修饰包括2’-甲氧乙氧基(2′-O-CH2CH2OCH3，也称为2’-O-(2-甲氧乙基)或2′-MOE)，即，烷氧基烷氧基基团。其它修饰包括2’-二甲基氨基氧基乙氧基，即O(CH2)2ON(CH3)2基团，也称为2′-DMAOE(如下文实施例中所述)，以及2’-二甲基氨基乙氧基乙氧基(本领域也称为2’-O-二甲基氨基乙氧基乙基或2′-DMAEOE)，即2′-O-CH2-O-CH2-N(CH2)2。Modified oligonucleotides may also comprise one or more substituted sugar moieties. Oligonucleotides contain one of the following at the 2' position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-, or N-alkynyl; or Oalkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl groups may be substituted or unsubstituted C to CO alkyl or C2 to CO alkenyl and alkynyl. In particular O(CH2)n OmCH3, O(CH2)n, OCH3, O(CH2)nNH2, O(CH2)nCH3, O(CH2)nONH2 and O(CH2nON(CH2)nCH3)2, where n and m can be 1 to about 10. Other oligonucleotides contain one of the following at the 2' position: C to CO, lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl, or O-aralkyl , SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, A substituted silyl group, an RNA cleavage group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents with similar properties. Modifications include 2'-methoxyethoxy (2'-O-CH2CH2OCH3, also known as 2'-O-(2-methoxyethyl) or 2'-MOE), ie, alkoxyalkoxy group. Other modifications include 2'-dimethylaminooxyethoxy, the O(CH2)2ON(CH3)2 group, also known as 2'-DMAOE (as described in the Examples below), and 2'- Dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethylaminoethoxyethyl or 2'-DMAEOE), ie 2'-O-CH2-O-CH2-N (CH2)2.
其它修饰包括2’-甲氧基(2′-O CH3)、2’-氨基丙氧基(2′-OCH2CH2CH2NH2)和2’-氟(2′-F)。类似的修饰也可在寡核苷酸的其它位置上进行，具体地在3’末端核苷酸上或2’-5’连接的寡核苷酸中糖的3’位置以及5’末端核苷酸的5’位置。寡核苷酸也可具有糖模拟物例如取代戊呋喃糖基糖的环丁基部分。教导制备这类经修饰的糖结构的代表性的美国专利包括但不限于，美国专利第4,981,957、5,118,800、5,319,080、5,359,044、5,393,878、5,446,137、5,466,786、5,514,785、5,519,134、5,567,811、5,576,427、5,591,722、5,597,909、5,610,300、5,627,053、5,639,873、5,646,265、5,658,873、5,670,633和5,700,920号，它们各自通过引用并入本文。Other modifications include 2'-methoxy (2'-O CH3), 2'-aminopropoxy (2'-OCH2CH2CH2NH2) and 2'-fluoro (2'-F). Similar modifications can also be made at other positions in the oligonucleotide, specifically the 3' position of the sugar on the 3' terminal nucleotide or in 2'-5' linked oligonucleotides and the 5' terminal nucleoside 5' position of the acid. Oligonucleotides may also have sugar mimetics such as cyclobutyl moieties substituted for pentofuranosyl sugars.教导制备这类经修饰的糖结构的代表性的美国专利包括但不限于，美国专利第4,981,957、5,118,800、5,319,080、5,359,044、5,393,878、5,446,137、5,466,786、5,514,785、5,519,134、5,567,811、5,576,427、5,591,722、5,597,909、5,610,300 , 5,627,053, 5,639,873, 5,646,265, 5,658,873, 5,670,633, and 5,700,920, each of which is incorporated herein by reference.
寡核苷酸也可包含核碱基(本领域常常简称为“碱基”)修饰或取代。本文所用的“未修饰的”或“天然的”核苷酸包括嘌呤碱基腺嘌呤(A)和鸟嘌呤(G)，以及嘧啶碱基胸腺嘧啶(T)、胞嘧啶(C)和尿嘧啶(U)。经修饰的核苷酸包括其它合成和天然核苷酸，例如5-甲基胞嘧啶(5-me-C)、5-羟甲基胞嘧啶、黄嘌呤、次黄嘌呤、2-氨基腺嘌呤、腺嘌呤和鸟嘌呤的6-甲基和其它烷基衍生物、腺嘌呤和鸟嘌呤的2-丙基和其它烷基衍生物、2-硫尿嘧啶、2-硫胸腺嘧啶和2-硫胞嘧啶、5-卤代尿嘧啶和胞嘧啶、5-丙炔基尿嘧啶和胞嘧啶、6-偶氮尿嘧啶、胞嘧啶和胸腺嘧啶、5-尿嘧啶(假尿嘧啶)、4-硫尿嘧啶、8-卤代、8-氨基、8-巯基、8-硫烷基、8-羟基和其它8-取代的腺嘌呤和鸟嘌呤、5-卤代具体地5-溴、5-三氟甲基和其它5-取代的尿嘧啶和胞嘧啶、7-甲基鸟嘌呤(methylquanine)和7-甲基腺嘌呤、8-氮杂鸟嘌呤和8-氮杂腺嘌呤、7-脱氮鸟嘌呤和7-脱氮腺嘌呤以及3-脱氮鸟嘌呤和3-脱氮腺嘌呤。Oligonucleotides may also contain nucleobase (often referred to in the art simply as "base") modifications or substitutions. As used herein, "unmodified" or "natural" nucleotides include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleotides include other synthetic and natural nucleotides such as 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine , 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thio Cytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, 6-azouracil, cytosine and thymine, 5-uracil (pseudouracil), 4-sulfur Uracil, 8-halo, 8-amino, 8-mercapto, 8-sulfanyl, 8-hydroxy and other 8-substituted adenine and guanine, 5-halo specifically 5-bromo, 5-tri Fluoromethyl and other 5-substituted uracil and cytosine, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deaza Guanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine.
此外，核苷酸包括公开于以下文献中的核苷酸：美国专利第3,687,808号、“TheConcise Encyclopedia of Polymer Science And Engineering”，858-859页，Kroschwitz，J.I.，编辑，John Wiley & Sons，1990、Englisch等，′Angewandle Chemie，International Edition′，1991，30，613页、和Sanghvi，Y.S.，第15章，“AntisenseResearch and Applications”，289-302页，Crooke，S.T.和Lebleu，B.ea.，CRC Press，1993。这些核苷酸中的某些对于增加本发明的寡聚化合物的结合亲和力特别地有用。这些包括5-取代嘧啶、6-氮杂嘧啶和N-2、N-6和0-6取代的嘌呤，包括2-氨丙基腺嘌呤、5-丙炔基尿嘧啶和5-丙炔基胞嘧啶。已显示5-甲基胞嘧啶取代增加核酸双链体的稳定性达0.6-1.2℃(Sanghvi，Y.S.，Crooke，S.T.和Lebleu，B.，编辑，“反义研究和应用”，CRC Press，BocaRaton，1993，276-278页)且为目前优选的碱基取代，甚至更特别地当与2’-O甲氧基乙基糖修饰组合时。In addition, nucleotides include those disclosed in U.S. Patent No. 3,687,808, "The Concise Encyclopedia of Polymer Science And Engineering", pp. 858-859, Kroschwitz, J.I., ed., John Wiley & Sons, 1990, Englisch et al., 'Angewandle Chemie, International Edition', 1991, 30, pp. 613, and Sanghvi, Y.S., Chapter 15, "Antisense Research and Applications", pp. 289-302, Crooke, S.T. and Lebleu, B.ea., CRC Press, 1993. Certain of these nucleotides are particularly useful for increasing the binding affinity of the oligomeric compounds of the invention. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines including 2-aminopropyladenine, 5-propynyluracil and 5-propynyl Cytosine. 5-Methylcytosine substitutions have been shown to increase the stability of nucleic acid duplexes up to 0.6-1.2°C (Sanghvi, Y.S., Crooke, S.T. and Lebleu, B., eds., "Antisense Research and Applications", CRC Press, Boca Raton , 1993, pp. 276-278) and is a presently preferred base substitution, even more particularly when combined with the 2'-Omethoxyethyl sugar modification.
教导制备上述经修饰的核苷酸以及其它经修饰的核苷酸的代表性的美国专利包括但不限于，美国专利第3,687,808、以及4,845,205、5,130,302、5,134,066、5,175,273、5,367,066、5,432,272、5,457,187、5,459,255、5,484,908、5,502,177、5,525,711、5,552,540、5,587,469、5,596,091、5,614,617、5,750,692和5,681,941号，它们各自通过引用并入本文。Representative U.S. patents that teach the preparation of the above-mentioned modified nucleotides, as well as other modified nucleotides, include, but are not limited to, U.S. Patent Nos. 5,484,908, 5,502,177, 5,525,711, 5,552,540, 5,587,469, 5,596,091, 5,614,617, 5,750,692, and 5,681,941, each of which is incorporated herein by reference.
本发明的寡核苷酸的另一种修饰涉及使所述寡核苷酸与一种或更多种部分或缀合物化学连接，该部分或缀合物增强所述寡核苷酸的活性、细胞分布或细胞摄取。Another modification of the oligonucleotides of the invention involves chemically linking the oligonucleotides to one or more moieties or conjugates that enhance the activity of the oligonucleotides , cellular distribution or cellular uptake.
这类部分包括但不限于：脂质部分，例如胆固醇部分、胆酸、硫醚例如己基-S-三苯甲基硫醇、硫代胆固醇、脂族链例如十二烷二醇或十一烷基残基、磷脂例如二-十六烷基-外消旋-甘油或1，2-二-O-十六烷基-外消旋-甘油-3-H-膦酸三乙铵、聚胺或聚乙二醇链、或金刚烷乙酸、棕榈基部分、或十八胺或己基氨基-羰基-t羟胆固醇部分。Such moieties include, but are not limited to: lipid moieties such as cholesterol moieties, cholic acids, thioethers such as hexyl-S-tritylthiol, thiocholesterol, aliphatic chains such as dodecanediol or undecane phospholipids such as di-hexadecyl-rac-glycerol or 1,2-di-O-hexadecyl-rac-glycerol-3-H-phosphonic acid triethylammonium, polyamine Or polyethylene glycol chains, or adamantaneacetic acid, palmityl moieties, or octadecylamine or hexylamino-carbonyl-t-hydroxycholesterol moieties.
教导制备这类寡核苷酸缀合物的代表性的美国专利包括但不限于，美国专利第4,828,979、4,948,882、5,218,105、5,525,465、5,541,313、5,545,730、5,552,538、5,578,717、5,580,731、5,580,731、5,591,584、5,109,124、5,118,802、5,138,045、5,414,077、5,486,603、5,512,439、5,578,718、5,608,046、4,587,044、4,605,735、4,667,025、4,762,779、4,789,737、4,824,941、4,835,263、4,876,335、4,904,582、4,958,013、5,082,830、5,112,963、5,214,136、5,082,830、5,112,963、5,214,136、5,245,022、5,254,469、5,258,506、5,262,536、5,272,250、5,292,873、5,317,098、5,371,241、5,391,723、5,416,203、5,451,463、5,510,475、5,512,667、5,514,785、5,565,552、5,567,810、5,574,142、5,585,481、5,587,371、5,595,726、5,597,696、5,599,923、5,599,928和5,688,941号，它们各自通过引用并入本文。教导制备这类寡核苷酸缀合物的代表性的美国专利包括但不限于，美国专利第4,828,979、4,948,882、5,218,105、5,525,465、5,541,313、5,545,730、5,552,538、5,578,717、5,580,731、5,580,731、5,591,584、5,109,124、5,118,802 、5,138,045、5,414,077、5,486,603、5,512,439、5,578,718、5,608,046、4,587,044、4,605,735、4,667,025、4,762,779、4,789,737、4,824,941、4,835,263、4,876,335、4,904,582、4,958,013、5,082,830、5,112,963、5,214,136、5,082,830、5,112,963、5,214,136、5,245,022、5,254,469、5,258,506 、5,262,536、5,272,250、5,292,873、5,317,098、5,371,241、5,391,723、5,416,203、5,451,463、5,510,475、5,512,667、5,514,785、5,565,552、5,567,810、5,574,142、5,585,481、5,587,371、5,595,726、5,597,696、5,599,923、5,599,928和5,688,941号，它们各自通过引用并入This article.
药物开发：本发明的化合物也可应用于药物开发和靶标验证的领域。本发明包括本文所鉴定的化合物和靶区段在阐明存在于抗病毒基因多核苷酸和疾病状态、表型或病况之间的关系的药物开发努力中的应用。这些方法包括检测或调节抗病毒基因多核苷酸，包括使本发明的化合物与样品、组织、细胞或生物体接触，在处理后的某个时间测定抗病毒基因多核苷酸的核酸或蛋白水平和/或相关的表型或化学终末点，以及任选将该测定值与未处理样品或与用本发明的另一种化合物处理的样品比较。这些方法也可与其它试验平行或组合进行以确定未知基因的功能用于靶标验证过程，或确定特定基因产物作为用于治疗或预防特定疾病、病况或表型的靶标的有效性。Drug development: The compounds of the present invention are also applicable in the fields of drug development and target validation. The present invention includes the use of the compounds and target segments identified herein in drug development efforts to elucidate the relationship that exists between an antiviral gene polynucleotide and a disease state, phenotype or condition. These methods include detecting or modulating antiviral gene polynucleotides, including contacting a compound of the invention with a sample, tissue, cell or organism, and measuring the nucleic acid or protein levels of the antiviral gene polynucleotides and and/or associated phenotypic or chemical endpoints, and optionally compare the assay to an untreated sample or to a sample treated with another compound of the invention. These methods can also be performed in parallel or in combination with other assays to determine the function of an unknown gene for use in the target validation process, or to determine the effectiveness of a particular gene product as a target for the treatment or prevention of a particular disease, condition or phenotype.
评价基因表达的增量调节或抑制：To evaluate up-regulation or inhibition of gene expression:
外源核酸到宿主细胞或生物体中的转移可通过直接检测细胞或生物体中核酸的存在情况来评价。这类检测可通过本领域众所周知的数种方法来完成。例如，外源核酸的存在情况可通过DNA印迹或通过聚合酶链式反应(PCR)技术使用以下引物来检测，该引物特异地扩增与所述核酸相关的核苷酸序列。外源核酸的表达也可使用包括基因表达分析在内的常规方法来测定。例如，由外源核酸产生的mRNA可使用RNA印迹和反转录PCR(RT-PCR)来检测和定量。Transfer of exogenous nucleic acid into a host cell or organism can be assessed by direct detection of the presence of the nucleic acid in the cell or organism. Such detection can be accomplished by several methods well known in the art. For example, the presence of exogenous nucleic acid can be detected by Southern blot or by polymerase chain reaction (PCR) techniques using primers that specifically amplify the nucleotide sequence associated with the nucleic acid. Expression of exogenous nucleic acid can also be determined using routine methods including gene expression analysis. For example, mRNA produced from exogenous nucleic acid can be detected and quantified using Northern blotting and reverse transcription PCR (RT-PCR).
来自外源核酸的RNA表达也可通过测定酶活性或报道蛋白活性来检测。例如，反义调节活性可根据靶核酸表达的减少或增加间接地测定，靶核酸表达作为外源核酸正在产生效应物RNA的指示。基于序列保守性，可设计和使用引物来扩增靶基因的编码区。最初，可使用来自每个基因的最高表达的编码区来建立模型对照基因，但任何编码或非编码区均可使用。每个对照基因通过将每个编码区插入报道基因编码区和其聚腺苷酸信号之间来装配。这些质粒可产生在基因的上游部分具有报道基因以及在3’非编码区中具有潜在RNAi靶标的mRNA。各个反义寡核苷酸的有效性可通过调节报道基因来测定。可用于本发明的方法中的报道基因包括乙酰羟酸合酶(AHAS)、碱性磷酸酶(AP)、β半乳糖苷酶(LacZ)、β葡糖醛酸酶(GUS)、氯霉素乙酰转移酶(CAT)、绿色荧光蛋白(GFP)、红色荧光蛋白(RFP)、黄色荧光蛋白(YFP)、青色荧光蛋白(CFP)、辣根过氧化物酶(HRP)、萤光素酶(Luc)、胭脂碱合酶(NOS)、章鱼碱合酶(OCS)，以及其衍生物。多重选择标记为可利用的，其赋予对氨苄青霉素、博来霉素、氯霉素、庆大霉素、潮霉素、卡那霉素、林可霉素、甲氨蝶呤、草丁膦(phosphinothricin)、嘌呤霉素和四环素的抗性。确定报道基因调节的方法为本领域众所周知的，包括但不限于，荧光法(例如荧光光谱法、荧光激活细胞分选术(FACS)、荧光显微法)、抗生素抗性测定。Expression of RNA from exogenous nucleic acid can also be detected by measuring enzyme activity or reporter protein activity. For example, antisense modulatory activity can be measured indirectly in terms of a decrease or increase in target nucleic acid expression as an indication that the exogenous nucleic acid is producing effector RNA. Based on sequence conservation, primers can be designed and used to amplify the coding region of the target gene. Initially, the most highly expressed coding region from each gene can be used to create a model control gene, but any coding or non-coding region can be used. Each control gene was assembled by inserting each coding region between the reporter gene coding region and its polyA signal. These plasmids produce mRNA with a reporter gene in the upstream portion of the gene and a potential RNAi target in the 3' non-coding region. The effectiveness of individual antisense oligonucleotides can be determined by modulating the reporter gene. Reporter genes that can be used in the methods of the invention include acetohydroxyacid synthase (AHAS), alkaline phosphatase (AP), beta galactosidase (LacZ), beta glucuronidase (GUS), chloramphenicol Acetyltransferase (CAT), green fluorescent protein (GFP), red fluorescent protein (RFP), yellow fluorescent protein (YFP), cyan fluorescent protein (CFP), horseradish peroxidase (HRP), luciferase ( Luc), nopaline synthase (NOS), octopine synthase (OCS), and derivatives thereof. Multiple selectable markers are available which confer resistance to ampicillin, bleomycin, chloramphenicol, gentamicin, hygromycin, kanamycin, lincomycin, methotrexate, glufosinate (phosphinothricin), puromycin and tetracycline resistance. Methods for determining reporter gene regulation are well known in the art and include, but are not limited to, fluorometric methods (eg, fluorescence spectroscopy, fluorescence activated cell sorting (FACS), fluorescence microscopy), antibiotic resistance assays.
RIG1、MDA5、IFNA1蛋白和mRNA表达可使用本领域技术人员已知和本文别处所描述的方法测定。例如，免疫测定法(例如ELISA)可用来测定蛋白水平。用于ELISA的抗病毒基因抗体可市购，例如，从R&D Systems(Minneapolis，MN)，Abcam，Cambridge，MA。RIG1, MDA5, IFNA1 protein and mRNA expression can be determined using methods known to those of skill in the art and described elsewhere herein. For example, immunoassays (eg, ELISA) can be used to measure protein levels. Antiviral genetic antibodies for use in ELISA are commercially available, eg, from R&D Systems (Minneapolis, MN), Abcam, Cambridge, MA.
在实施方案中，使用本发明反义寡核苷酸处理的样品(例如，体内或体外细胞或组织)中的RIG1、MDA5、IFNA1表达(例如，mRNA或蛋白)通过与对照样品中的抗病毒基因表达相比较来评价。例如，蛋白或核酸表达可使用本领域技术人员已知的方法，与模拟处理或未处理样品中的蛋白或核酸表达相比较。或者，与用对照反义寡核苷酸(例如，具有已改变或不同序列的反义寡核苷酸)处理的样品的比较可根据所需信息来进行。在另一个实施方案中，可将已处理样品对比未处理样品在抗病毒基因蛋白或核酸表达方面的差异，与已处理样品对比未处理样品在不同核酸(包括研究者认为适当的任何标准，例如，持家基因)表达方面的差异相比较。In an embodiment, RIG1, MDA5, IFNA1 expression (for example, mRNA or protein) in a sample (for example, in vivo or in vitro cells or tissues) treated with an antisense oligonucleotide of the present invention is compared with antiviral expression in a control sample. Gene expression was evaluated in comparison. For example, protein or nucleic acid expression can be compared to protein or nucleic acid expression in mock-treated or untreated samples using methods known to those skilled in the art. Alternatively, comparisons to samples treated with control antisense oligonucleotides (eg, antisense oligonucleotides with altered or different sequences) can be made depending on the desired information. In another embodiment, the difference in the antiviral gene protein or nucleic acid expression of the treated sample can be compared with the untreated sample, and the difference in the different nucleic acid (including any standard that the researcher thinks is appropriate, such as , housekeeping genes) expression differences were compared.
可将观察到的差异根据需要例如以比例或分数的形式表达，用于与对照的比较。在实施方案中，抗病毒基因mRNA或蛋白水平，在用本发明反义寡核苷酸处理的样品中，相对于未处理样品或用对照核酸处理的样品增加至约1.25倍-约10倍或更多或减少至约1/1.25-约1/10或更少。在实施方案中，抗病毒基因mRNA或蛋白水平增加至至少约1.25倍、至少约1.3倍、至少约1.4倍、至少约1.5倍、至少约1.6倍、至少约1.7倍、至少约1.8倍、至少约2倍、至少约2.5倍、至少约3倍、至少约3.5倍、至少约4倍、至少约4.5倍、至少约5倍、至少约5.5倍、至少约6倍、至少约6.5倍、至少约7倍、至少约7.5倍、至少约8倍、至少约8.5倍、至少约9倍、至少约9.5倍、或至少约10倍或更多，或减少至至多约1/1.25、至多约1/1.3、至多约1/1.4、至多约1/1.5、至多约1/1.6、至多约1/1.7、至多约1/1.8、至多约1/2、至多约1/2.5、至多约1/3、至多约1/3.5、至多约1/4、至多约1/4.5、至多约1/5、至多约1/5.5、至多约1/6、至多约1/6.5、至多约1/7、至多约1/7.5、至多约1/8、至多约1/8.5、至多约1/9、至多约1/9.5、或至多约1/10或更少。Observed differences can be expressed as desired, eg, as a ratio or fraction, for comparison to controls. In embodiments, antiviral gene mRNA or protein levels, in samples treated with antisense oligonucleotides of the present invention, are increased to about 1.25-fold to about 10-fold relative to untreated samples or samples treated with control nucleic acids or More or reduced to about 1/1.25 to about 1/10 or less. In embodiments, antiviral gene mRNA or protein levels are increased by at least about 1.25-fold, at least about 1.3-fold, at least about 1.4-fold, at least about 1.5-fold, at least about 1.6-fold, at least about 1.7-fold, at least about 1.8-fold, at least about 2 times, at least about 2.5 times, at least about 3 times, at least about 3.5 times, at least about 4 times, at least about 4.5 times, at least about 5 times, at least about 5.5 times, at least about 6 times, at least about 6.5 times, at least About 7 times, at least about 7.5 times, at least about 8 times, at least about 8.5 times, at least about 9 times, at least about 9.5 times, or at least about 10 times or more, or reduced to at most about 1/1.25, at most about 1 /1.3, at most about 1/1.4, at most about 1/1.5, at most about 1/1.6, at most about 1/1.7, at most about 1/1.8, at most about 1/2, at most about 1/2.5, at most about 1/3 , up to about 1/3.5, up to about 1/4, up to about 1/4.5, up to about 1/5, up to about 1/5.5, up to about 1/6, up to about 1/6.5, up to about 1/7, up to About 1/7.5, at most about 1/8, at most about 1/8.5, at most about 1/9, at most about 1/9.5, or at most about 1/10 or less.
试剂盒、研究试剂、诊断和治疗Kits, Research Reagents, Diagnostics and Therapeutics
本发明的化合物可用于诊断、治疗和预防，并作为研究试剂和试剂盒的组分。此外，能够灵敏特异地抑制基因表达的反义寡核苷酸，常常被普通技术人员用于阐明特定基因的功能或区分生物途径的各个成员之间的功能。The compounds of the invention are useful in diagnosis, therapy and prophylaxis, and as research reagents and components of kits. In addition, antisense oligonucleotides, which can sensitively and specifically inhibit gene expression, are often used by those of ordinary skill to elucidate the function of a specific gene or to distinguish the function between various members of a biological pathway.
对于用于试剂盒和诊断和各种生物系统，本发明的化合物(单独的或与其它化合物或治疗剂组合)可用作在差异和/或组合分析中的工具，以阐明在细胞和组织内表达的基因的一部分或全部互补序列的表达模式。For use in kits and diagnostics and various biological systems, the compounds of the invention (alone or in combination with other compounds or therapeutic agents) can be used as tools in differential and/or combinatorial analysis to elucidate the The expression pattern of a portion or the entire complement of an expressed gene.
本文所用的术语“生物系统”或“系统”定义为表达或使得能够表达抗病毒基因产物的任何生物体、细胞、细胞培养物或组织。这些包括但不限于人、转基因动物、细胞、细胞培养物、组织、异种移植物、移植物及其组合。The term "biological system" or "system" as used herein is defined as any organism, cell, cell culture or tissue that expresses or enables the expression of an antiviral gene product. These include, but are not limited to, humans, transgenic animals, cells, cell cultures, tissues, xenografts, grafts, and combinations thereof.
作为一个非限制性实例，将在用一种或更多种反义化合物处理的细胞或组织内的表达模式与未用反义化合物处理的对照细胞或组织相比较，并针对基因表达的差异水平分析产生的模式，因为它们有关于，例如，所检测基因的疾病相关、信号传导途径、细胞定位、表达水平、大小、结构或功能。这些分析可对受刺激或未受刺激的细胞以及在影响表达模式的其它化合物存在或不存在时进行。As a non-limiting example, expression patterns in cells or tissues treated with one or more antisense compounds are compared to control cells or tissues not treated with antisense compounds, and differential levels of gene expression are targeted The resulting patterns are analyzed as they relate to, for example, disease association, signaling pathway, cellular localization, expression level, size, structure or function of the detected gene. These assays can be performed on stimulated or unstimulated cells and in the presence or absence of other compounds that affect expression patterns.
本领域已知的基因表达分析方法的实例包括DNA阵列或微阵列(Brazma和Vilo，(2000)FEBS Lett.，480，17-24；Celis等，(2000)FEBS Lett.，480，2-16)、SAGE(基因表达的系列分析)(Madden等，(2000)Drug Discov.Today，5，415-425)、READS(已消化cDNA的限制酶扩增)(Prashar和Weissman，(1999)Methods Enzymol.，303，258-72)、TOGA(总基因表达分析)(Sutcliffe等，(2000)Proc.Natl.Acad.Sci.U.S.A.，97，1976-81)、蛋白质阵列和蛋白质组学(Celis等，(2000)FEBS Lett.，480，2-16；Jungblut等，Electrophoresis，1999，20，2100-10)、已表达序列标志(EST)测序(Celis等，FEBS Lett.，2000，480，2-16；Larsson等，J.Biotechnol.，2000，80，143-57)、消减(subtractive)RNA指纹法(SuRF)(Fuchs等，(2000)Anal.Biochem.286，91-98；Larson等，(2000)Cytometry 41，203-208)、消减克隆、差异显示(DD)(Jurecic和Belmont，(2000)Curr.Opin.Microbiol.3，316-21)、比较基因组杂交(Carulli等，(1998)J.Cell Biochem.Suppl.，31，286-96)、FISH(荧光原位杂交)技术(Going和Gusterson，(1999)Eur.J.Cancer，35，1895-904)和质谱分析法(To，Comb.(2000)Chem.High Throughput Screen，3，235-41)。Examples of gene expression analysis methods known in the art include DNA arrays or microarrays (Brazma and Vilo, (2000) FEBS Lett., 480, 17-24; Celis et al., (2000) FEBS Lett., 480, 2-16 ), SAGE (serial analysis of gene expression) (Madden et al., (2000) Drug Discov. Today, 5, 415-425), READS (restriction enzyme amplification of digested cDNA) (Prashar and Weissman, (1999) Methods Enzymol ., 303, 258-72), TOGA (Total Gene Expression Analysis) (Sutcliffe et al., (2000) Proc.Natl.Acad.Sci.U.S.A., 97, 1976-81), protein array and proteomics (Celis et al., (2000) FEBS Lett., 480, 2-16; Jungblut et al., Electrophoresis, 1999, 20, 2100-10), expressed sequence tag (EST) sequencing (Celis et al., FEBS Lett., 2000, 480, 2-16 Larsson et al., J.Biotechnol., 2000,80,143-57), subtractive (subtractive) RNA fingerprinting (SuRF) (Fuchs et al., (2000) Anal.Biochem.286,91-98; Larson et al., (2000 ) Cytometry 41, 203-208), subtractive cloning, differential display (DD) (Jurecic and Belmont, (2000) Curr.Opin.Microbiol.3, 316-21), comparative genomic hybridization (Carulli et al., (1998) J. Cell Biochem.Suppl., 31,286-96), FISH (fluorescence in situ hybridization) technology (Going and Gusterson, (1999) Eur.J.Cancer, 35,1895-904) and mass spectrometry (To, Comb. (2000) Chem. High Throughput Screen, 3, 235-41).
本发明的化合物对于研究和诊断而言为有用的，因为这些化合物与编码抗病毒基因的核酸杂交。例如，作为有效的抗病毒基因调节剂以本文公开的这类效率和这类条件下杂交的寡核苷酸，在有利于基因扩增或检测的条件下分别为有效的引物或探针。这些引物和探针可用于需要对编码抗病毒基因的核酸分子特异性的检测的方法中，和可用于扩增所述核酸分子，以用于检测或用于进一步研究抗病毒基因。本发明的反义寡核苷酸(具体地，引物和探针)与编码抗病毒基因的核酸的杂交，可通过本领域已知的方法来检测。这类方法可包括使酶与所述寡核苷酸缀合、放射性标记所述寡核苷酸或任何其它适当的检测方法。也可制备使用这类检测方法来检测样品中抗病毒基因水平的试剂盒。The compounds of the invention are useful for research and diagnostics because these compounds hybridize to nucleic acids encoding antiviral genes. For example, an oligonucleotide that hybridizes with such efficiencies and under such conditions disclosed herein as an effective antiviral gene modulator is an effective primer or probe under conditions favorable for gene amplification or detection, respectively. These primers and probes can be used in methods requiring specific detection of nucleic acid molecules encoding antiviral genes, and can be used to amplify said nucleic acid molecules for detection or for further study of antiviral genes. The hybridization of antisense oligonucleotides of the present invention (specifically, primers and probes) to nucleic acids encoding antiviral genes can be detected by methods known in the art. Such methods may include conjugating an enzyme to the oligonucleotide, radiolabeling the oligonucleotide, or any other suitable method of detection. Kits for detecting levels of antiviral genes in samples using such detection methods can also be prepared.
反义物的特异性和灵敏性也由本领域技术人员掌握用于治疗用途。已将反义化合物在动物(包括人)的疾病状态的治疗中用作治疗部分。反义寡核苷酸药物已安全和有效地施用给人且许多临床试验目前正在进行。因此确立的是，反义化合物可为有用的治疗形式，可将其经配置以在用于治疗细胞、组织和动物、尤其是人的治疗方案中有用。The specificity and sensitivity of antisense for therapeutic use is also within the grasp of those skilled in the art. Antisense compounds have been used as therapeutic moieties in the treatment of disease states in animals, including humans. Antisense oligonucleotide drugs have been safely and effectively administered to humans and many clinical trials are currently underway. It is thus established that antisense compounds can be useful therapeutic forms that can be formulated for use in therapeutic regimens for the treatment of cells, tissues and animals, especially humans.
对于治疗而言，将怀疑具有可通过调节抗病毒基因多核苷酸的表达来治疗的疾病或病症的动物(优选人)，通过施用根据本发明的反义化合物来治疗。例如，在一个非限制性实施方案中，所述方法包括：给需要治疗的动物施用治疗上有效量的抗病毒基因调节剂的步骤。本发明的抗病毒基因调节剂有效地调节抗病毒基因的活性或调节抗病毒基因蛋白的表达。在一个实施方案中，动物中抗病毒基因的活性或表达与对照相比抑制了约10％。优选地，将动物中抗病毒基因的活性或表达抑制约30％。更优选地，将动物中抗病毒基因的活性或表达抑制50％或更多。因此，与对照相比，寡聚化合物将抗病毒基因mRNA的表达调节至少10％、至少50％、至少25％、至少30％、至少40％、至少50％、至少60％、至少70％、至少75％、至少80％、至少85％、至少90％、至少95％、至少98％、至少99％、或100％。For treatment, an animal, preferably a human, suspected of having a disease or condition treatable by modulating the expression of an antiviral gene polynucleotide is treated by administering an antisense compound according to the invention. For example, in one non-limiting embodiment, the method includes the step of administering to the animal in need of treatment a therapeutically effective amount of an antiviral gene modulator. The antiviral gene regulator of the present invention can effectively regulate the activity of antiviral gene or regulate the expression of antiviral gene protein. In one embodiment, the activity or expression of the antiviral gene is inhibited in the animal by about 10% compared to a control. Preferably, the activity or expression of the antiviral gene in the animal is inhibited by about 30%. More preferably, the activity or expression of the antiviral gene in the animal is inhibited by 50% or more. Thus, the oligomeric compound modulates the expression of antiviral gene mRNA by at least 10%, at least 50%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, At least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%.
在一个实施方案中，与对照相比，在动物中抗病毒基因的活性或表达增加约10％。优选地，在动物中抗病毒基因的活性或表达增加约30％。更优选地，在动物中抗病毒基因的活性或表达增加50％或更多。因此，与对照比较，寡聚化合物使抗病毒基因mRNA的表达调节至少10％、至少50％、至少25％、至少30％、至少40％、至少50％、至少60％、至少70％、至少75％、至少80％、至少85％、至少90％、至少95％、至少98％、至少99％或100％。In one embodiment, the activity or expression of the antiviral gene is increased by about 10% in the animal compared to a control. Preferably, the activity or expression of the antiviral gene is increased by about 30% in the animal. More preferably, the activity or expression of the antiviral gene is increased by 50% or more in the animal. Thus, the oligomeric compound modulates the expression of antiviral gene mRNA by at least 10%, at least 50%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%.
例如，抗病毒基因表达的下降可在动物的血清、血液、脂肪组织、肝脏或任何其它体液、组织或器官中测定。优选地，包含于待分析的所述液体、组织或器官之内的细胞包含编码抗病毒基因肽的核酸分子和/或抗病毒基因蛋白本身。For example, a decrease in expression of an antiviral gene can be measured in the animal's serum, blood, adipose tissue, liver or any other body fluid, tissue or organ. Preferably, cells contained within said fluid, tissue or organ to be analyzed comprise nucleic acid molecules encoding antiviral gene peptides and/or antiviral gene proteins themselves.
本发明的化合物可通过向合适的药学上可接受的稀释剂或载体中添加有效量的化合物来用于药物组合物。本发明的化合物和方法的应用也可为预防上有用的。The compounds of the present invention can be used in pharmaceutical compositions by adding an effective amount of the compound to a suitable pharmaceutically acceptable diluent or carrier. Use of the compounds and methods of the invention may also be useful prophylactically.
缀合物：本发明的寡核苷酸的另一种修饰涉及以化学方法将一种或更多种增强寡核苷酸的活性、细胞分布或细胞摄取的部分或缀合物与寡核苷酸连接。这些部分或缀合物可包含与官能团(例如伯羟基或仲羟基)共价结合的缀合基团。本发明的缀合基团包括嵌入剂、报道分子、聚胺、聚酰胺、聚乙二醇、聚醚、增强寡聚体药效学特性的基团，以及增强寡聚体药代动力学特性的基团。典型的缀合基团包括胆固醇、脂质、磷脂、生物素、吩嗪、叶酸、菲啶、蒽醌、吖啶、荧光黄、罗丹明、香豆素和染料。增强药效学特性的基团，在本发明的背景中，包括改善摄取、增强对降解的抗性和/或加强与靶核酸的序列特异性杂交的基团。增强药代动力学特性的基团，在本发明的背景中，包括改善本发明的化合物的摄取、分布、代谢或分泌的基团。代表性的缀合基团在提交于1992年10月23日的国际专利申请第PCT/US92/09196号和美国专利第6,287,860号中公开，所述文献通过引用并入本文。缀合部分包括但不限于，脂质部分(例如胆固醇部分)、胆酸、硫醚(例如，己基-5-三苯甲基硫醇)、硫代胆固醇、脂族链(例如，十二烷二醇或十一烷基残基)、磷脂(例如，二-十六烷基-外消旋-甘油或1，2-二-O-十六烷基-外消旋-甘油-3-H-膦酸三乙铵)、聚胺或聚乙二醇链、或金刚烷乙酸、棕榈基部分、或十八胺或己基氨基-羰基-羟胆固醇部分。也可将本发明的寡核苷酸与活性药物物质缀合，例如，阿司匹林、华法林、保泰松、布洛芬、舒洛芬、芬布芬、酮洛芬、(S)-(+)普拉洛芬、卡洛芬、丹酰肌氨酸、2，3，5-三碘苯甲酸、氟芬那酸、亚叶酸、苯并噻二嗪、氯噻嗪、二氮杂吲哚美辛(indomethicin)、巴比妥酸盐、头孢菌素、磺胺类药物、抗糖尿病药、抗菌剂或抗生素。Conjugates: Another modification of the oligonucleotides of the invention involves chemically combining one or more moieties or conjugates that enhance the activity, cellular distribution, or cellular uptake of the oligonucleotide with the oligonucleotide acid connection. These moieties or conjugates may comprise conjugating groups covalently bonded to functional groups such as primary or secondary hydroxyl groups. Conjugate groups of the invention include intercalators, reporters, polyamines, polyamides, polyethylene glycols, polyethers, groups that enhance the pharmacodynamic properties of oligomers, and groups that enhance oligomer pharmacokinetic properties group. Typical conjugation groups include cholesterol, lipids, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluorescein, rhodamine, coumarin, and dyes. Groups that enhance pharmacodynamic properties include, in the context of the present invention, groups that improve uptake, increase resistance to degradation and/or enhance sequence-specific hybridization to target nucleic acids. Groups that enhance the pharmacokinetic properties, in the context of the present invention, include groups that improve the uptake, distribution, metabolism or secretion of the compounds of the invention. Representative conjugate groups are disclosed in International Patent Application No. PCT/US92/09196, filed October 23, 1992, and US Patent No. 6,287,860, which are incorporated herein by reference. Conjugation moieties include, but are not limited to, lipid moieties (e.g., cholesterol moieties), cholic acids, thioethers (e.g., hexyl-5-tritylthiol), thiocholesterol, aliphatic chains (e.g., dodecane diol or undecyl residues), phospholipids (e.g., di-hexadecyl-rac-glycerol or 1,2-di-O-hexadecyl-rac-glycerol-3-H - triethylammonium phosphonate), polyamine or polyethylene glycol chains, or adamantaneacetic acid, palmityl moieties, or octadecylamine or hexylamino-carbonyl-hydroxycholesterol moieties. The oligonucleotides of the invention can also be conjugated to active drug substances, for example, aspirin, warfarin, phenylbutazone, ibuprofen, suprofen, fenbufen, ketoprofen, (S)-(+) Pranoprofen, carprofen, dansylsarcosine, 2,3,5-triiodobenzoic acid, flufenamic acid, leucovorin, benzothiadiazine, chlorothiazide, diazepine Indomethicin, barbiturates, cephalosporins, sulfonamides, antidiabetics, antibacterials, or antibiotics.
教导制备这类寡核苷酸缀合物的代表性的美国专利包括但不限于，美国专利第4,828,979、4,948,882、5,218,105、5,525,465、5,541,313、5,545,730、5,552,538、5,578,717、5,580,731、5,580,731、5,591,584、5,109,124、5,118,802、5,138,045、5,414,077、5,486,603、5,512,439、5,578,718、5,608,046、4,587,044、4,605,735、4,667,025、4,762,779、4,789,737、4,824,941、4,835,263、4,876,335、4,904,582、4,958,013、5,082,830、5,112,963、5,214,136、5,082,830、5,112,963、5,214,136、5,245,022、5,254,469、5,258,506、5,262,536、5,272,250、5,292,873、5,317,098、5,371,241、5,391,723、5,416,203、5,451,463、5,510,475、5,512,667、5,514,785、5,565,552、5,567,810、5,574,142、5,585,481、5,587,371、5,595,726、5,597,696、5,599,923、5,599,928和5,688,941号。教导制备这类寡核苷酸缀合物的代表性的美国专利包括但不限于，美国专利第4,828,979、4,948,882、5,218,105、5,525,465、5,541,313、5,545,730、5,552,538、5,578,717、5,580,731、5,580,731、5,591,584、5,109,124、5,118,802 、5,138,045、5,414,077、5,486,603、5,512,439、5,578,718、5,608,046、4,587,044、4,605,735、4,667,025、4,762,779、4,789,737、4,824,941、4,835,263、4,876,335、4,904,582、4,958,013、5,082,830、5,112,963、5,214,136、5,082,830、5,112,963、5,214,136、5,245,022、5,254,469、5,258,506 、5,262,536、5,272,250、5,292,873、5,317,098、5,371,241、5,391,723、5,416,203、5,451,463、5,510,475、5,512,667、5,514,785、5,565,552、5,567,810、5,574,142、5,585,481、5,587,371、5,595,726、5,597,696、5,599,923、5,599,928和5,688,941号。
制剂：本发明的化合物也可与其它分子、分子结构或化合物的混合物混合、封装、缀合或以其它方式缔合，作为例如，脂质体、受体靶向分子、口服的、直肠的、局部的或其它制剂，用于有助于摄取、分布和/或吸收。教导制备这类摄取、分布和/或吸收辅助制剂的代表性的美国专利包括但不限于，美国专利第5,108,921、5,354,844、5,416,016、5,459,127、5,521,291、5,543,165、5,547,932、5,583,020、5,591,721、4,426,330、4,534,899、5,013,556、5,108,921、5,213,804、5,227,170、5,264,221、5,356,633、5,395,619、5,416,016、5,417,978、5,462,854、5,469,854、5,512,295、5,527,528、5,534,259、5,543,152、5,556,948、5,580,575和5,595,756号，它们各自通过引用并入本文。Formulations: Compounds of the invention may also be mixed, encapsulated, conjugated or otherwise associated with other molecules, molecular structures or mixtures of compounds, as, for example, liposomes, receptor targeting molecules, oral, rectal, Topical or other formulations used to facilitate uptake, distribution and/or absorption. Representative U.S. patents that teach the preparation of such uptake, distribution, and/or absorption aiding formulations include, but are not limited to, U.S. Patent Nos. 、5,108,921、5,213,804、5,227,170、5,264,221、5,356,633、5,395,619、5,416,016、5,417,978、5,462,854、5,469,854、5,512,295、5,527,528、5,534,259、5,543,152、5,556,948、5,580,575和5,595,756号，它们各自通过引用并入本文。
尽管，反义寡核苷酸不需要在载体的情况中施用以便调节靶表达和/或功能，但是本发明的实施方案涉及用于反义寡核苷酸表达的表达载体构建体，包括启动子、杂合启动子基因序列并且拥有强组成型启动子活性，或可在所需情况下诱导的启动子活性。Although, antisense oligonucleotides need not be administered in the context of a vector in order to modulate target expression and/or function, embodiments of the invention relate to expression vector constructs for expression of antisense oligonucleotides, including promoters , a hybrid promoter gene sequence and possesses strong constitutive promoter activity, or a promoter activity that can be induced under desired conditions.
在一个实施方案中，本发明实施涉及用适合的核酸递送系统施用至少一种前述反义寡核苷酸。在一个实施方案中，该系统包含与多核苷酸可操作连接的非病毒载体。这类非病毒载体的实例包括单独的寡核苷酸(例如，SEQ ID NO：10-30中的任何一个或更多个)或与适合的蛋白、多糖或脂质制剂组合的寡核苷酸。In one embodiment, practice of the invention involves administering at least one of the aforementioned antisense oligonucleotides with a suitable nucleic acid delivery system. In one embodiment, the system comprises a non-viral vector operably linked to a polynucleotide. Examples of such non-viral vectors include oligonucleotides (e.g., any one or more of SEQ ID NOs: 10-30) alone or in combination with suitable protein, polysaccharide or lipid formulations .
其它适合的核酸递送系统包括病毒载体，典型地来自腺病毒、腺病毒伴随病毒(AAV)、依赖辅助病毒的腺病毒、逆转录病毒或仙台病毒-脂质体(HVJ)复合体中的至少一种的序列。优选地，所述病毒载体包含与多核苷酸可操作连接的强真核启动子，例如，巨细胞病毒(CMV)启动子。Other suitable nucleic acid delivery systems include viral vectors, typically from at least one of an adenovirus, adeno-associated virus (AAV), helper-dependent adenovirus, retrovirus, or Sendai virus-liposome (HVJ) complex. species sequence. Preferably, the viral vector comprises a strong eukaryotic promoter, eg, a cytomegalovirus (CMV) promoter, operably linked to the polynucleotide.
另外，载体包括病毒载体、融合蛋白和化学缀合物。逆转录病毒载体包括莫洛尼鼠白血病病毒和基于HIV的病毒。一种基于HIV的病毒载体包括至少两种载体，其中gag基因和pol基因来自HIV基因组而env基因来自另一种病毒。DNA病毒载体为优选的。这些载体包括痘病毒载体(例如正痘病毒或禽痘病毒载体)、疱疹病毒载体(例如单纯疱疹I病毒(HSV)载体、腺病毒载体和腺伴随病毒载体)。Additionally, vectors include viral vectors, fusion proteins and chemical conjugates. Retroviral vectors include Moloney murine leukemia virus and HIV-based viruses. An HIV-based viral vector includes at least two vectors in which the gag and pol genes are from the HIV genome and the env gene is from another virus. DNA viral vectors are preferred. These vectors include poxvirus vectors (eg, orthopoxvirus or fowlpoxvirus vectors), herpesvirus vectors (eg, herpes simplex I virus (HSV) vectors, adenovirus vectors, and adeno-associated virus vectors).
本发明的反义化合物包括任何药学上可接受的盐、酯、或这类酯的盐、或任何其它化合物，其在施用给动物包括人后，能够提供(直接地或间接地)生物学活性的代谢物或其残留物。Antisense compounds of the present invention include any pharmaceutically acceptable salts, esters, or salts of such esters, or any other compound that, when administered to animals, including humans, is capable of conferring (directly or indirectly) biological activity metabolites or their residues.
术语“药学上可接受的盐”是指本发明化合物的生理上和药学上可接受的盐：即，保留母体化合物的所需生物活性且不对其赋予非所需的毒理学作用的盐。对于寡核苷酸而言，药学上可接受的盐的实例和其使用进一步描述于美国专利第6,287,860号中，其通过引用并入本文。The term "pharmaceutically acceptable salt" refers to a physiologically and pharmaceutically acceptable salt of a compound of the invention: ie, a salt that retains the desired biological activity of the parent compound and does not impart undesired toxicological effects thereto. For oligonucleotides, examples of pharmaceutically acceptable salts and their uses are further described in US Patent No. 6,287,860, which is incorporated herein by reference.
本发明也包括包含本发明的反义化合物的药物组合物和制剂。本发明的药物组合物可以以若干方式来施用，这取决于是否需要局部或全身治疗以及待治疗的区域。施用可为局部的(包括眼的和至黏膜包括阴道和直肠递送)、肺的(例如，通过吸入或吹入散剂或气雾剂，包括通过喷雾器)、气管内的、鼻内的、表皮的和经皮的、口服的或胃肠外的。胃肠外施用包括静脉内、动脉内、皮下、腹膜内或肌肉注射或输注；或颅内(例如，鞘内或心室内)施用。The invention also includes pharmaceutical compositions and formulations comprising the antisense compounds of the invention. The pharmaceutical compositions of the invention can be administered in several ways, depending on whether local or systemic treatment is desired and the area to be treated. Administration can be topical (including ophthalmic and to mucous membranes including vaginal and rectal delivery), pulmonary (for example, by inhalation or insufflation of powder or aerosol, including by nebulizer), intratracheal, intranasal, epidermal and transdermal, oral or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial (eg, intrathecal or intraventricular) administration.
对于治疗中枢神经系统中的组织而言，可通过例如注射或输注进入脑脊髓液进行施用。施用反义RNA进入脑脊髓液已在例如美国专利申请公开第2007/0117772号，“Methodsfor slowing familial ALS disease progression”中描述，该申请通过引用整体并入本文。For treatment of tissues in the central nervous system, administration can be by, for example, injection or infusion into the cerebrospinal fluid. Administration of antisense RNA into cerebrospinal fluid has been described, for example, in US Patent Application Publication No. 2007/0117772, "Methods for slowing familial ALS disease progression," which is hereby incorporated by reference in its entirety.
如果意图将本发明的反义寡核苷酸施用给中枢神经系统中的细胞，可与一种或更多种能够促进主题反义寡核苷酸渗透穿过血脑屏障的物质一起施用。注射可在例如内嗅皮质或海马中进行。通过施用腺病毒载体递送神经营养因子至肌肉组织中的运动神经元描述于，例如，美国专利第6,632,427号，“Adenoviral-vector-mediated gene transfer intomedullary motor neurons”，其通过引用并入本文。直接递送载体至脑(例如，纹状体、丘脑、海马或黑质)为本领域已知且描述于例如美国专利第6,756,523号，“Adenovirusvectors for the transfer of foreign genes into cells of the central nervoussystem particularly in brain”，其通过引用并入本文。施用可快速，如通过注射，或在一段时间内进行，如通过缓慢输注或施用缓释制剂。If it is intended to administer the antisense oligonucleotides of the invention to cells in the central nervous system, it may be administered with one or more substances capable of facilitating penetration of the subject antisense oligonucleotides across the blood-brain barrier. Injections can be made, for example, in the entorhinal cortex or hippocampus. Delivery of neurotrophic factors to motor neurons in muscle tissue by administration of adenoviral vectors is described, for example, in US Patent No. 6,632,427, "Adenoviral-vector-mediated gene transfer into medullary motor neurons," which is incorporated herein by reference. Direct delivery of vectors to the brain (e.g., striatum, thalamus, hippocampus, or substantia nigra) is known in the art and described, for example, in U.S. Pat. No. 6,756,523, "Adenovirus vectors for the transfer of foreign genes into cells of the central nervous system particularly in brain", which is incorporated herein by reference. Administration can be rapid, such as by injection, or over a period of time, such as by slow infusion or administration of a sustained release formulation.
主题反义寡核苷酸也可与提供了所需药学或药效学特性的物质连接或缀合。例如，反义寡核苷酸可与本领域已知的促进渗透或转运穿过血脑屏障的任何物质(例如转铁蛋白受体的抗体)偶联，并通过静脉注射施用。反义化合物可与病毒载体连接，例如，使反义化合物更有效和/或增加反义化合物转运穿过血脑屏障的病毒载体。渗透性血脑屏障破坏也可通过例如输注糖或氨基酸来完成，所述糖包括但不限于，内消旋赤藓醇、木糖醇、D(+)半乳糖、D(+)乳糖、D(+)木糖、卫矛醇、肌醇、L(-)果糖、D(-)甘露醇、D(+)葡萄糖、D(+)阿拉伯糖、D(-)阿拉伯糖、纤维二糖、D(+)麦芽糖、D(+)蜜三糖、L(+)鼠李糖、D(+)蜜二糖、D(-)核糖、侧金盏糖醇、D(+)阿拉伯糖醇、L(-)阿拉伯糖醇、D(+)岩藻糖、L(-)岩藻糖、D(-)来苏糖、L(+)来苏糖和L(-)来苏糖，所述氨基酸包括但不限于，谷氨酰胺、赖氨酸、精氨酸、天冬酰胺、天冬氨酸、半胱氨酸、谷氨酸、甘氨酸、组氨酸、亮氨酸、甲硫氨酸、苯丙氨酸、脯氨酸、丝氨酸、苏氨酸、酪氨酸、缬氨酸和牛磺酸。用于增强血脑屏障渗透的方法和材料描述于，例如，美国专利第4,866,042号，“Method for the delivery of genetic material acrossthe blood brain barrier”，第6,294,520号，“Material for passage through theblood-brain barrier”，和第6,936,589号，“Parenteral delivery systems”，全部通过引用整体并入本文。The subject antisense oligonucleotides may also be linked or conjugated to substances that provide the desired pharmaceutical or pharmacodynamic properties. For example, antisense oligonucleotides can be conjugated to any substance known in the art to facilitate penetration or transport across the blood-brain barrier (eg, antibodies to the transferrin receptor) and administered intravenously. An antisense compound can be linked to a viral vector, eg, a viral vector that renders the antisense compound more effective and/or increases translocation of the antisense compound across the blood-brain barrier. Permeable blood-brain barrier disruption can also be accomplished, for example, by infusion of sugars or amino acids, including, but not limited to, mesoerythritol, xylitol, D(+)galactose, D(+)lactose, D(+) xylose, dulcitol, inositol, L(-) fructose, D(-) mannitol, D(+) glucose, D(+) arabinose, D(-) arabinose, cellobiose , D(+) maltose, D(+) raffinose, L(+) rhamnose, D(+) melibiose, D(-) ribose, side calendrol, D(+) arabitol , L(-) arabitol, D(+) fucose, L(-) fucose, D(-) lyxose, L(+) lyxose and L(-) lyxose, all Such amino acids include, but are not limited to, glutamine, lysine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glycine, histidine, leucine, methionine acid, phenylalanine, proline, serine, threonine, tyrosine, valine and taurine. Methods and materials for enhancing blood-brain barrier penetration are described, for example, in U.S. Patent No. 4,866,042, "Method for the delivery of genetic material across the blood brain barrier," and U.S. Patent No. 6,294,520, "Material for passage through the blood-brain barrier" , and No. 6,936,589, "Parenteral delivery systems," all incorporated herein by reference in their entirety.
主题反义化合物也可与其它分子、分子结构或化合物的混合物混合、封装、缀合或以其它方式缔合，作为例如，脂质体、受体靶向分子、口服的、直肠的、局部的或其它制剂，用于有助于摄取、分布和/或吸收。例如，阳离子脂质可包含在制剂中以促进寡核苷酸摄取。一种显示出促进摄取的这类组合物为LIPOFECTIN(可从GIBCO-BRL，Bethesda，MD获得)。The subject antisense compounds can also be mixed, encapsulated, conjugated or otherwise associated with other molecules, molecular structures or mixtures of compounds as, for example, liposomes, receptor targeting molecules, oral, rectal, topical or other formulations to facilitate uptake, distribution and/or absorption. For example, cationic lipids can be included in the formulation to facilitate oligonucleotide uptake. One such composition shown to enhance uptake is LIPOFECTIN (available from GIBCO-BRL, Bethesda, MD).
认为带有至少一个2’-O-甲氧基乙基修饰的寡核苷酸对于口服施用而言为特别有用的。用于局部施用的药物组合物和制剂可包括透皮贴剂、软膏剂、洗剂、乳膏剂、凝胶剂、滴剂、栓剂、喷雾剂、液体和散剂。常规药物载体、水性、粉末或油性基质、增稠剂等可为必要的或所需的。包被的避孕套、手套等也可为有用的。Oligonucleotides bearing at least one 2'-O-methoxyethyl modification are believed to be particularly useful for oral administration. Pharmaceutical compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable. Coated condoms, gloves, etc. may also be useful.
可适宜地以单位剂型存在的本发明药物制剂，可根据药学工业中众所周知的常规技术来制备。这类技术包括使活性成分与药物载体或赋形剂组合的步骤。一般而言，制剂如下制备：通过使活性成分与液体载体或细碎的固体载体或两者均匀和紧密地组合，随后在需要时使产物成形。The pharmaceutical formulations of the invention, conveniently presented in unit dosage form, may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredient with a pharmaceutical carrier or excipient. In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.
可将本发明的组合物制成任何许多可能剂型，例如但不限于，片剂、胶囊剂、凝胶胶囊、液体糖浆、软凝胶、栓剂和灌肠剂。也可将本发明的组合物在水性、非水性或混合介质中制成混悬剂。水性混悬剂可进一步包含增加混悬剂粘度的物质，包括例如羧甲基纤维素钠、山梨醇和/或葡聚糖。所述混悬剂也可包含稳定剂。Compositions of the present invention may be formulated in any of a number of possible dosage forms such as, but not limited to, tablets, capsules, gel capsules, liquid syrups, soft gels, suppositories and enemas. The compositions of the present invention may also be prepared as suspensions in aqueous, non-aqueous or mixed media. Aqueous suspensions may further contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol, and/or dextran. The suspension may also contain stabilizers.
本发明的药物组合物包括但不限于，溶液剂、乳剂、泡沫剂和含脂质体的制剂。本发明的药物组合物和制剂可包含一种或更多种渗透促进剂、载体、赋形剂或其它活性或非活性成分。Pharmaceutical compositions of the present invention include, but are not limited to, solutions, emulsions, foams, and liposome-containing formulations. The pharmaceutical compositions and formulations of the invention may contain one or more penetration enhancers, carriers, excipients or other active or inactive ingredients.
乳剂典型地为一种液体以通常直径超过0.1μm的液滴形式分散在另一种液体中的非均质体系。乳剂可包含除分散相之外的其它组分，以及可作为在水相、油相中的溶液或其本身作为单独相存在的活性药物。将微乳液包括为本发明的一个实施方案。乳剂及其使用为本领域众所周知的且进一步描述于美国专利第6,287,860号中。Emulsions are typically heterogeneous systems in which one liquid is dispersed in another liquid in the form of droplets usually exceeding 0.1 [mu]m in diameter. Emulsions may contain other components than the dispersed phase, and the active drug may be present as a solution in an aqueous phase, an oily phase, or itself as a separate phase. The inclusion of microemulsions is one embodiment of the invention. Emulsions and their use are well known in the art and are further described in US Patent No. 6,287,860.
本发明的制剂包括脂质体制剂。本发明所用的术语“脂质体”意为由排列在一个或复数个球形双层中的两亲脂质组成的囊泡。脂质体为具有由亲脂材料形成的膜和包含待递送组合物的水性内部的单层或多层囊泡。阳离子脂质体为带正电的脂质体，认为其与带负电的DNA分子相互作用以形成稳定的复合体。认为pH敏感的或带负电的脂质体诱捕DNA而不是与其复合。阳离子和非阳离子脂质体均已用来递送DNA到细胞。Formulations of the invention include liposomal formulations. The term "liposome" as used herein means a vesicle consisting of amphiphilic lipids arranged in one or more spherical bilayers. Liposomes are unilamellar or multilamellar vesicles having a membrane formed of lipophilic material and an aqueous interior containing the composition to be delivered. Cationic liposomes are positively charged liposomes that are believed to interact with negatively charged DNA molecules to form stable complexes. pH-sensitive or negatively charged liposomes are thought to entrap DNA rather than complex it. Both cationic and non-cationic liposomes have been used to deliver DNA to cells.
脂质体也包括“空间上稳定的”脂质体，该术语如本文所用是指包含一种或更多种特化脂质的脂质体。当掺入到脂质体中时，这些特化脂质给脂质体带来相对于缺乏这类特化脂质的脂质体增长的循环生命期。空间上稳定的脂质体的实例为以下脂质体，其中脂质体形成囊泡的脂质部分的部分包含一种或更多种糖脂或用一种或更多种亲水聚合物，例如聚乙二醇(PEG)部分衍生化。脂质体及其使用进一步描述于美国专利第6,287,860号中。Liposomes also include "sterically stable" liposomes, which term as used herein refers to liposomes comprising one or more specialized lipids. When incorporated into liposomes, these specialized lipids confer to liposomes an increased circulating lifetime relative to liposomes lacking such specialized lipids. Examples of sterically stable liposomes are liposomes in which part of the vesicle-forming lipid portion of the liposome comprises one or more glycolipids or with one or more hydrophilic polymers, For example polyethylene glycol (PEG) partial derivatization. Liposomes and their use are further described in US Patent No. 6,287,860.
本发明的药物制剂和组合物也可包含表面活性剂。表面活性剂在药物产品、制剂和乳剂中的使用为本领域众所周知的。表面活性剂及其使用进一步描述于美国专利第6,287,860号中，其通过引用并入本文。The pharmaceutical formulations and compositions of the invention may also contain surfactants. The use of surfactants in pharmaceutical products, formulations and emulsions is well known in the art. Surfactants and their use are further described in US Patent No. 6,287,860, which is incorporated herein by reference.
在一个实施方案中，本发明使用各种渗透促进剂来实现核酸特别是寡核苷酸的有效递送。除了有助于非亲脂性药物穿过细胞膜的扩散之外，渗透促进剂还增加亲脂性药物的渗透性。可将渗透促进剂归类为属于五大类的一种，五大类即表面活性剂、脂肪酸、胆汁盐、螯合剂和非螯合非表面活性剂。渗透促进剂及其使用进一步描述于美国专利第6,287,860号，其通过引用并入本文。In one embodiment, the present invention employs various penetration enhancers to achieve efficient delivery of nucleic acids, particularly oligonucleotides. In addition to facilitating the diffusion of non-lipophilic drugs across cell membranes, penetration enhancers also increase the permeability of lipophilic drugs. Penetration enhancers can be classified as belonging to one of five broad classes, namely surfactants, fatty acids, bile salts, chelating agents and non-chelating non-surfactants. Penetration enhancers and their use are further described in US Patent No. 6,287,860, which is incorporated herein by reference.
本领域的技术人员将认识到，根据其预期用途(即给药途径)来常规地设计制剂。Those skilled in the art will recognize that formulations are routinely designed according to their intended use (ie, route of administration).
用于局部给药的制剂包括以下制剂，其中本发明寡核苷酸与局部递送剂(例如，脂质、脂质体、脂肪酸、脂肪酸酯、甾类化合物、螯合剂和表面活性剂)混合。脂质和脂质体包括中性的(例如二油酰基-磷脂酰DOPE乙醇胺、二肉豆蔻酰基磷脂酰胆碱DMPC、二硬脂酰基磷脂酰胆碱)、阴性的(例如二肉豆蔻酰基磷脂酰甘油DMPG)和阳离子的(例如二油酰基四甲基氨丙基DOTAP和二油酰基-磷脂酰基乙醇胺DOTMA)。Formulations for topical administration include formulations wherein oligonucleotides of the invention are admixed with topical delivery agents such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents, and surfactants. . Lipids and liposomes include neutral (e.g. dioleoyl-phosphatidyl DOPE ethanolamine, dimyristoylphosphatidylcholine DMPC, distearoylphosphatidylcholine), negative (e.g. dimyristoylphosphatidylcholine acylglycerol DMPG) and cationic (eg dioleoyl tetramethylaminopropyl DOTAP and dioleoyl-phosphatidylethanolamine DOTMA).
对于局部或其它给药而言，可将本发明的寡核苷酸封装在脂质体内或可与其(特别是与阳离子脂质体)形成复合体。或者，可将寡核苷酸与脂质(特别是阳离子脂质)复合。脂肪酸和酯类、其药学上可接受的盐以及它们的使用进一步描述于美国专利第6,287,860号中。For topical or other administration, the oligonucleotides of the invention may be encapsulated within liposomes or may form complexes therewith, especially with cationic liposomes. Alternatively, oligonucleotides can be complexed to lipids, particularly cationic lipids. Fatty acids and esters, their pharmaceutically acceptable salts, and their use are further described in US Patent No. 6,287,860.
用于口服给药的组合物和制剂包括散剂或颗粒剂、微粒、纳米粒子、在水或非水性介质中的混悬剂或溶液剂、胶囊剂、凝胶胶囊、小药囊、片剂或小片。增稠剂、矫味剂、稀释剂、乳化剂、分散助剂或粘合剂可为所需的。口服制剂为以下制剂，其中将本发明的寡核苷酸与一种或更多种渗透促进剂、表面活性剂和螯合剂联合施用。表面活性剂包括脂肪酸和/或其酯或盐、胆汁酸和/或其盐。胆汁酸/盐和脂肪酸及其使用进一步描述于美国专利第6,287,860号中，其通过引用并入本文。还有渗透促进剂的组合，例如脂肪酸/盐与胆汁酸/盐的组合。一种特别的组合为月桂酸的钠盐、癸酸和UDCA。另外的渗透促进剂包括聚氧化乙烯-9-月桂醚、聚氧化乙烯-20-鲸蜡醚。本发明的寡核苷酸可以以包括喷雾干燥颗粒的颗粒形式口服地递送，或络合以形成微米或纳米粒子。寡核苷酸络合剂及其使用进一步描述于美国专利第6,287,860号中，其通过引用并入本文。Compositions and formulations for oral administration include powders or granules, microparticles, nanoparticles, suspensions or solutions in water or non-aqueous media, capsules, gel capsules, sachets, tablets or small pieces. Thickeners, flavoring agents, diluents, emulsifiers, dispersion aids or binders may be desired. Oral formulations are those wherein the oligonucleotides of the invention are administered in combination with one or more penetration enhancers, surfactants and chelating agents. Surfactants include fatty acids and/or esters or salts thereof, bile acids and/or salts thereof. Bile acids/salts and fatty acids and their use are further described in US Patent No. 6,287,860, which is incorporated herein by reference. There are also combinations of penetration enhancers, such as combinations of fatty acids/salts and bile acids/salts. A particular combination is the sodium salt of lauric acid, capric acid and UDCA. Additional penetration enhancers include polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether. The oligonucleotides of the invention can be delivered orally in particulate form, including spray-dried particles, or complexed to form micro- or nanoparticles. Oligonucleotide complexing agents and their use are further described in US Patent No. 6,287,860, which is incorporated herein by reference.
用于胃肠外、鞘内或心室内给药的组合物和制剂可包括无菌水性溶液剂，其也可含有缓冲液、稀释剂和其它适合的添加剂，例如但不限于，渗透促进剂、载体化合物和其它药学上可接受的载体或赋形剂。Compositions and formulations for parenteral, intrathecal or intraventricular administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives such as, but not limited to, penetration enhancers, Carrier compounds and other pharmaceutically acceptable carriers or excipients.
本发明的某些实施方案提供了药物组合物，所述药物组合物包含一种或更多种寡聚化合物和一种或更多种其它通过非反义机制来起作用的化学治疗剂。这类化学治疗剂的实例包括但不限于癌症化学治疗药物，例如柔红霉素、道诺霉素、更生霉素、多柔比星、表柔比星、伊达比星、依索比星、博来霉素、马磷酰胺、异环磷酰胺、胞嘧啶阿拉伯糖苷、双氯乙基-亚硝基脲、白消安、丝裂霉素C、放线菌素D、光神霉素、泼尼松、羟孕酮、睾酮、他莫昔芬、达卡巴嗪、丙卡巴肼、六甲蜜胺、五甲蜜胺、米托蒽醌、安吖啶、苯丁酸氮芥、甲基环己基亚硝基脲、氮芥、美法仑、环磷酰胺、6-巯基嘌呤、6-巯鸟嘌呤、阿糖胞苷、5-氮杂胞苷、羟基脲、喷司他丁、4-羟基过氧环磷酰胺、5-氟尿嘧啶(5-FU)、5-氟脱氧尿苷(5-FUdR)、甲氨蝶呤(MTX)、秋水仙碱、泰素、长春新碱、长春碱、依托泊苷(VP-16)、三甲曲沙、伊立替康、拓泊替康、吉西他滨、替尼泊苷、顺铂和己烯雌酚(DES)。当与本发明的化合物一起使用时，这类化学治疗剂可单独地(例如，5-FU和寡核苷酸)、序贯地(例如，5-FU和寡核苷酸持续一段时间，接着MTX和寡核苷酸)、或与一种或更多种其它的这类化学治疗剂组合(例如，5-FU、MTX和寡核苷酸，或5-FU、放射疗法和寡核苷酸)使用。抗炎药(包括但不限于非甾体抗炎药和皮质类固醇)和抗病毒药物(包括但不限于利巴韦林(ribivirin)、阿糖腺苷、阿昔洛韦和更昔洛韦)也可组合到本发明的组合物中。反义化合物和其它非反义药物的组合也在本发明的范围之内。两种或更多种组合的化合物可一起或序贯使用。Certain embodiments of the invention provide pharmaceutical compositions comprising one or more oligomeric compounds and one or more other chemotherapeutic agents that act through a non-antisense mechanism. Examples of such chemotherapeutic agents include, but are not limited to, cancer chemotherapeutic drugs such as daunorubicin, daunorubicin, dactinomycin, doxorubicin, epirubicin, idarubicin, esorubicin , Bleomycin, Maphosphamide, Ifosfamide, Cytosine Arabinoside, Dichloroethyl-Nitrosourea, Busulfan, Mitomycin C, Actinomycin D, Mithramycin , prednisone, hydroxyprogesterone, testosterone, tamoxifen, dacarbazine, procarbazine, hexamethylmelamine, pentamethylmelamine, mitoxantrone, amsacrine, chlorambucil, methyl Cyclohexylnitrosourea, nitrogen mustard, melphalan, cyclophosphamide, 6-mercaptopurine, 6-mercaptoguanine, cytarabine, 5-azacytidine, hydroxyurea, pentostatin, 4 -Hydroxycyclophosphamide, 5-fluorouracil (5-FU), 5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX), colchicine, taxol, vincristine, vinblastine , etoposide (VP-16), trimetrexate, irinotecan, topotecan, gemcitabine, teniposide, cisplatin, and diethylstilbestrol (DES). When used with the compounds of the invention, such chemotherapeutic agents can be used alone (e.g., 5-FU and oligonucleotide), sequentially (e.g., 5-FU and oligonucleotide for a period of time, followed by MTX and oligonucleotides), or in combination with one or more other such chemotherapeutic agents (for example, 5-FU, MTX and oligonucleotides, or 5-FU, radiation therapy and oligonucleotides )use. Anti-inflammatory drugs (including but not limited to NSAIDs and corticosteroids) and antiviral drugs (including but not limited to ribavirin, vidarabine, acyclovir, and ganciclovir) It can also be incorporated into the compositions of the present invention. Combinations of antisense compounds and other non-antisense drugs are also within the scope of the invention. Two or more compounds in combination may be used together or sequentially.
在另一个相关的实施方案中，本发明的组合物可包含靶向第一核酸的一种或更多种反义化合物(特别是寡核苷酸)，以及靶向第二核酸靶标的一种或更多种其它反义化合物。例如，第一靶标可为抗病毒基因的特定反义序列，第二靶标可为来自另一个核苷酸序列的区域。或者，本发明的组合物可包含靶向相同抗病毒基因核酸靶标的不同区域的两种或更多种反义化合物。本文举例说明了许多反义化合物的实例而其它的可选自本领域已知的适合化合物。两种或更多种组合化合物可一起或序贯使用。In another related embodiment, the compositions of the invention may comprise one or more antisense compounds (particularly oligonucleotides) targeted to a first nucleic acid, and one or more antisense compounds targeted to a second nucleic acid target. or more other antisense compounds. For example, the first target may be a specific antisense sequence of an antiviral gene and the second target may be a region from another nucleotide sequence. Alternatively, a composition of the invention may comprise two or more antisense compounds targeting different regions of the same antiviral gene nucleic acid target. A number of examples of antisense compounds are illustrated herein and others may be selected from suitable compounds known in the art. Two or more combination compounds may be used together or sequentially.
给药：Administration:
认为治疗组合物的制剂和其随后的施用(给药)在本领域技术人员的技术之内。给药取决于要治疗的疾病状态的严重性和应答性，而疗程从数天持续到数月，或直到完成治愈或达到疾病状态的减轻。最佳给药方案可根据患者体内药物蓄积的测定来计算。普通技术人员可容易地确定最适剂量、给药方法和重复率。最适剂量可根据单独寡核苷酸的相对功效而不同，一般可基于发现在体外和体内动物模型中有效的EC50来评价。一般而言，剂量为0.01μg-100g/kg体重，且可每天、每周、每月或每年给药一次或更多次，或甚至每2-20年一次。本领域普通技术人员可基于测定体液或组织中药物的停留时间和浓度来容易地评估给药的重复率。成功治疗之后，可能需要使患者进行维持疗法以预防疾病状态的复发，其中所述寡核苷酸以维持剂量来施用，范围为0.01μg-100g/kg体重，每天一次或更多次到每20年一次。The formulation of therapeutic compositions and their subsequent administration (administration) are considered to be within the skill of those skilled in the art. Dosing depends on the severity and responsiveness of the disease state being treated, and the course of treatment can be from days to months, or until cure is achieved or remission of the disease state is achieved. The optimal dosing regimen can be calculated based on the determination of drug accumulation in the patient's body. One of ordinary skill can readily determine optimum dosages, dosing methodologies and repetition rates. Optimal dosages may vary according to the relative potencies of the individual oligonucleotides and can generally be evaluated based on EC50s found to be effective in in vitro and in vivo animal models. In general, dosages will range from 0.01 μg to 100 g/kg body weight, and may be administered one or more times daily, weekly, monthly or yearly, or even once every 2-20 years. One of ordinary skill in the art can readily assess the repetition rate of dosing based on determining the residence time and concentration of the drug in bodily fluids or tissues. Following successful treatment, it may be desirable to subject the patient to maintenance therapy to prevent recurrence of the disease state, wherein the oligonucleotide is administered at a maintenance dose ranging from 0.01 μg to 100 g/kg body weight, once or more per day to every 20 once a year.
在实施方案中，使用下列剂量的药物治疗患者，所述剂量为至少约1、至少约2、至少约3、至少约4、至少约5、至少约6、至少约7、至少约8、至少约9、至少约10、至少约15、至少约20、至少约25、至少约30、至少约35、至少约40、至少约45、至少约50、至少约60、至少约70、至少约80、至少约90、或至少约100mg/kg体重。反义寡核苷酸的某些注射剂量描述于，例如，美国专利第7,563,884号，“Antisense modulation of PTP1B expression”，通过引用以其整体并入本文。In an embodiment, the patient is treated with a dose of at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least About 9, at least about 10, at least about 15, at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, at least about 50, at least about 60, at least about 70, at least about 80 , at least about 90, or at least about 100 mg/kg body weight. Certain injectable doses of antisense oligonucleotides are described, for example, in US Patent No. 7,563,884, "Antisense modulation of PTP1B expression," which is incorporated herein by reference in its entirety.
虽然上文已描述了本发明的各种实施方案，但是应理解的是，其仅以实例的方式提供，而并非限制。对公开的实施方案的许多改变可根据本文的公开内容来进行，而不会背离本发明的精神或范围。因此，本发明的广度和范围不应受到任何上述的实施方案所限制。While various embodiments of the present invention have been described above, it should be understood that they have been presented by way of example only, and not limitation. Many changes to the disclosed embodiments may be made in light of the disclosure herein without departing from the spirit or scope of the invention. Thus, the breadth and scope of the present invention should not be limited by any of the above-described embodiments.
本文提及的所有文献都通过引用结合到本文中。本申请引用的所有出版物和专利文献都为所有目的而通过引用结合，引用程度如同单独地指出各个单独出版物或专利文件一样。至于申请人在本文件中对不同参考文献的引用，申请人并不承认任何具体参考文献对其发明而言为“现有技术”。本发明的组合物和方法的实施方案举例说明于下列实施例中。All documents mentioned herein are hereby incorporated by reference. All publications and patent documents cited in this application are incorporated by reference for all purposes to the same extent as if each individual publication or patent document was individually indicated. With regard to Applicant's citations of various references in this document, Applicant does not admit that any particular reference is "prior art" with respect to its invention. Embodiments of the compositions and methods of the present invention are illustrated in the following examples.
实施例Example
下列非限制性的实施例用于举例说明本发明的经挑选的实施方案。应理解的是，所示组分的比例变化和要素备选对本领域的技术人员而言为显而易见的且在本发明的实施方案的范围之内。The following non-limiting examples serve to illustrate selected embodiments of the invention. It is to be understood that variations in the proportions of the components shown and substitutions of elements will be apparent to those skilled in the art and are within the scope of the embodiments of the invention.
实施例1：对抗病毒基因的反义核酸分子/或抗病毒基因多核苷酸有义链特异性的反义寡核苷酸的设计Embodiment 1: the design of the antisense nucleic acid molecule of antiviral gene/or the antisense oligonucleotide of antiviral gene polynucleotide sense strand specificity
如上指出的术语“对……特异性的寡核苷酸”或“靶向……的寡核苷酸”是指具有以下序列的寡核苷酸，该序列(i)能够与被靶向的基因的一部分形成稳定的复合体，或(ii)能够与被靶向的基因的mRNA转录物的一部分形成稳定的双链体。The term "oligonucleotide specific for ..." or "oligonucleotide targeting ..." as indicated above refers to an oligonucleotide having a sequence (i) capable of interacting with the targeted A portion of the gene forms a stable complex, or (ii) is capable of forming a stable duplex with a portion of the mRNA transcript of the gene being targeted.
适当寡核苷酸的挑选通过使用计算机程序来促进，该计算机程序自动比对核酸序列并指出同一性或同源性区域。这类程序用于比较例如通过搜索诸如GenBank等数据库或通过测序PCR产物而获得的核酸序列。来自一系列物种的核酸序列的比较允许挑选显示出物种之间适当同一性程度的核酸序列。在未测序基因的情况下，进行DNA印迹来确定在靶物种和其它物种的基因之间的同一性程度。通过在不同严格性程度下进行DNA印迹，如本领域众所周知的，可获得同一性的近似测量。这些程序允许挑选这样的寡核苷酸，其对在待控制的受试者中的靶核酸序列表现高度的互补性而对在其它物种中的相应核酸序列表现较低程度的互补性。本领域技术人员将认识到，在挑选用于本发明的适当的基因区域上具有相当大的自由。Selection of appropriate oligonucleotides is facilitated by the use of computer programs that automatically align nucleic acid sequences and indicate regions of identity or homology. Such programs are used to compare nucleic acid sequences obtained, for example, by searching databases such as GenBank or by sequencing PCR products. Comparison of nucleic acid sequences from a range of species allows the selection of nucleic acid sequences showing an appropriate degree of identity between species. In the case of unsequenced genes, Southern blots are performed to determine the degree of identity between genes of the target species and other species. Approximate measures of identity can be obtained by performing Southern blots at varying degrees of stringency, as is well known in the art. These procedures allow the selection of oligonucleotides that exhibit a high degree of complementarity to the target nucleic acid sequence in the subject to be controlled and a lower degree of complementarity to the corresponding nucleic acid sequence in other species. Those skilled in the art will recognize that there is considerable freedom in selecting appropriate gene regions for use in the present invention.
反义化合物为“可特异性地杂交的”，如果所述化合物与靶核酸的结合干扰靶核酸的正常功能，导致功能和/或活性的调节，并且在要求特异性结合的条件下具有足够程度的互补性以避免所述反义化合物与非靶核酸序列的非特异性结合，所述条件即在体内测定或治疗处理情况中的生理条件下，以及其中测定在体外测定情况中进行的条件下。An antisense compound is "specifically hybridizable" if the binding of the compound to the target nucleic acid interferes with the normal function of the target nucleic acid, resulting in modulation of function and/or activity, and to a sufficient extent under conditions requiring specific binding complementarity to avoid non-specific binding of the antisense compound to non-target nucleic acid sequences, ie under physiological conditions in the case of in vivo assays or therapeutic treatments, and in the case of in vitro assays.
本文所述的寡核苷酸的杂交特性可通过本领域已知的一种或更多种体外测定法来确定。例如，本文所述的寡核苷酸的特性可通过使用解链曲线测定法确定靶天然反义物和潜在药物分子之间的结合强度来获得。The hybridization properties of the oligonucleotides described herein can be determined by one or more in vitro assays known in the art. For example, the properties of the oligonucleotides described herein can be obtained by determining the strength of binding between a target natural antisense and a potential drug molecule using a melting curve assay.
靶天然反义物和潜在药物分子(Molecule)之间的结合强度，可使用任何已建立的测定分子间相互作用强度的方法例如解链曲线测定法来评价。The binding strength between the target natural antisense and the potential drug molecule (Molecule) can be assessed using any established method for measuring the strength of intermolecular interactions such as melting curve assays.
解链曲线测定法确定这样的温度，在该温度下天然反义物/Molecule复合体发生从双链构象到单链构象的迅速转变。此温度被广泛认可为两个分子之间相互作用强度的可靠衡量。Melting curve assays determine the temperature at which the natural antisense/Molecule complex undergoes a rapid transition from a double-stranded to a single-stranded conformation. This temperature is widely accepted as a reliable measure of the strength of the interaction between two molecules.
解链曲线测定法可使用实际的天然反义RNA分子的cDNA拷贝或对应Molecule的结合位点的合成DNA或RNA核苷酸来进行。包含进行此测定的所有必需试剂的多种试剂盒为可得的(例如Applied Biosystems Inc.MeltDoctor试剂盒)。这些试剂盒包含含有双链DNA(dsDNA)结合染料(例如ABI HRM染料、SYBR Green、SYTO，等等)之一的适宜的缓冲溶液。dsDNA染料的特性为，其在游离形式几乎不发出荧光，但当与dsDNA结合时为高度荧光的。Melting curve assays can be performed using cDNA copies of actual natural antisense RNA molecules or synthetic DNA or RNA nucleotides corresponding to the Molecule's binding site. A variety of kits are available containing all the necessary reagents to perform this assay (eg Applied Biosystems Inc. MeltDoctor kit). These kits contain a suitable buffered solution containing one of the double-stranded DNA (dsDNA) binding dyes (eg, ABI HRM dyes, SYBR Green, SYTO, etc.). A property of dsDNA dyes is that they emit little fluorescence in free form, but are highly fluorescent when bound to dsDNA.
为进行所述测定，将所述cDNA或相应寡核苷酸以由具体制造商的方案限定的浓度与Molecule混合。将所述混合物加热到95℃以解离所有预先形成的dsDNA复合体，然后缓慢冷却到室温或由试剂盒制造商确定的其它较低的温度以使DNA分子退火。随后将新形成的复合体缓慢加热到95℃，同时连续地收集由反应产生的荧光量的数据。荧光强度反比于反应中存在的dsDNA量。数据可使用与所述试剂盒相配的实时PCR仪器(例如ABI的StepOnePlus Real Time PCR系统或LightTyper仪器，Roche Diagnostics，Lewes，UK)来收集。To perform the assay, the cDNA or corresponding oligonucleotides are mixed with Molecules at concentrations defined by the specific manufacturer's protocol. The mixture is heated to 95°C to dissociate any pre-formed dsDNA complexes, then slowly cooled to room temperature or other lower temperature as determined by the kit manufacturer to anneal the DNA molecules. The newly formed complex was then slowly heated to 95°C while continuously collecting data on the amount of fluorescence produced by the reaction. Fluorescence intensity is inversely proportional to the amount of dsDNA present in the reaction. Data can be collected using a real-time PCR instrument compatible with the kit (eg, ABI's StepOnePlus Real Time PCR system or LightTyper instrument, Roche Diagnostics, Lewes, UK).
解链峰通过使用适当软件(例如LightTyper(Roche)或SDS Dissociation Curve，ABI)针对温度(x-轴)绘制荧光关于温度的负导数(在y-轴上的-d(荧光)/dT)的图形来构建。分析数据以确定从dsDNA复合体迅速转变到单链分子的温度。此温度称为Tm且正比于两个分子之间的相互作用强度。典型地，Tm将超过40℃。Melting peaks are determined by graphing the negative derivative of fluorescence with respect to temperature (-d(fluorescence)/dT on the y-axis) against temperature (x-axis) using appropriate software (e.g. LightTyper (Roche) or SDS Dissociation Curve, ABI). Construct. Data were analyzed to determine the temperature at which the rapid transition from dsDNA complexes to single-stranded molecules occurs. This temperature is called Tm and is proportional to the strength of interaction between two molecules. Typically, the Tm will exceed 40°C.
实施例2：抗病毒基因多核苷酸的调节Embodiment 2: the regulation of antiviral gene polynucleotide
用反义寡核苷酸处理HepG2细胞Treatment of HepG2 cells with antisense oligonucleotides
使来自ATCC的HepG2细胞(目录号HB-8065)在37℃和5％CO2下生长于生长培养基(MEM/EBSS(Hyclone目录号SH30024或Mediatech目录号MT-10-010-CV)+10％FBS(Mediatech目录号MT35-011-CV)+青霉素/链霉素(Mediatech目录号MT30-002-CI))。在试验的前一天，将所述细胞以密度1.5×105/ml再接种到6孔板，并在37℃和5％CO2下培养。在试验当天，将6孔板中的培养基换成新鲜的生长培养基。将所有反义寡核苷酸稀释到20μM的浓度。将2μl此溶液与400μl Opti-MEM培养基(Gibco目录号31985-070)和4μl脂质体转染胺2000(Invitrogen目录号11668019)在室温温育20min，然后施用到具有HepG2细胞的6孔板的每个孔。含有2μl水(代替所述寡核苷酸溶液)的类似混合物用于假转染的对照。在37℃和5％CO2下培养3-18h后，将培养基换成新鲜的生长培养基。添加反义寡核苷酸48h后，将培养基移出，并遵循制造商的说明书，使用Promega的SV全RNA分离系统(目录号Z3105)或Qiagen的RNeasy全RNA分离试剂盒(目录号74181)从细胞中提取RNA。如制造商的方案中所述，向使用Thermo Scientific的Verso cDNA试剂盒(目录号AB 1453B)或高容量cDNA反转录试剂盒(目录号4368813)进行的反转录反应中添加600ng RNA。将来自此反转录反应的cDNA用于通过实时PCR监测基因表达，其中使用ABI Taqman Gene Expression Mix(目录号4369510)和由ABI(Applied Biosystems Taqman Gene Expression Assay：Hs00204833_m1、Hs00223420_m1、Hs00855471_g1，Applied Biosystems Inc.，Foster City CA)设计的引物/探针。使用下面的PCR循环：50℃，2min；95℃，10min；40个(95℃，15秒；60℃，1min)循环，使用Mx4000热循环仪(Stratagene)。HepG2 cells from ATCC (Catalog No. HB-8065) were grown at 37°C and 5%CO in Growth Medium (MEM/EBSS (Hyclone Cat. No. SH30024 or Mediatech Cat. No. MT-10-010-CV) + 10 % FBS (Mediatech Cat. No. MT35-011-CV) + Penicillin/Streptomycin (Mediatech Cat. No. MT30-002-CI)). The day before the experiment, the cells were reseeded into 6-well plates at a density of 1.5×105 /ml and cultured at 37° C. and 5% CO2 . On the day of the experiment, the medium in the 6-well plate was replaced with fresh growth medium. All antisense oligonucleotides were diluted to a concentration of 20 μM. 2 μl of this solution was incubated with 400 μl of Opti-MEM medium (Gibco Cat. No. 31985-070) and 4 μl of Lipofectamine 2000 (Invitrogen Cat. No. 11668019) for 20 min at room temperature and then applied to a 6-well plate with HepG2 cells each hole. A similar mixture containing 2 μl of water (instead of the oligonucleotide solution) was used as a mock-transfected control. After 3-18 h of incubation at 37 °C and 5%CO , the medium was replaced with fresh growth medium. 48h after the addition of antisense oligonucleotides, the medium was removed, and following the manufacturer’s instructions, Promega’s SV Total RNA Isolation System (Cat. No. Z3105) or Qiagen’s RNeasy Total RNA Isolation Kit (Cat. No. 74181) was used to extract Extract RNA from cells. 600 ng of RNA was added to a reverse transcription reaction performed using Thermo Scientific's Verso cDNA Kit (Cat# AB 1453B) or High Capacity cDNA Reverse Transcription Kit (Cat# 4368813) as described in the manufacturer's protocol. The cDNA from this reverse transcription reaction was used to monitor gene expression by real-time PCR using the ABI Taqman Gene Expression Mix (Catalog No. 4369510) and by ABI (Applied Biosystems Taqman Gene Expression Assay: Hs00204833_m1, Hs00223420_m1, Hs00855471_g1, Applied Biosystems Inc ., Foster City CA) designed primers/probes. The following PCR cycle was used: 50°C, 2 min; 95°C, 10 min; 40 (95°C, 15 sec; 60°C, 1 min) cycles using a Mx4000 thermal cycler (Stratagene).
基于处理的和假转染的样品之间18S-标准化的dCt值的不同，计算用反义寡核苷酸处理后基因表达的倍数变化。Fold changes in gene expression after treatment with antisense oligonucleotides were calculated based on the difference in 18S-normalized dCt values between treated and mock-transfected samples.
结果result
实时PCR结果表明，在用针对RIG1反义物hs.601664和AK300104的siRNA处理后48h，HepG2细胞中RIG1mRNA的水平显著增加(图1)。Real-time PCR results showed that the level of RIG1 mRNA in HepG2 cells was significantly increased 48 h after treatment with siRNA against RIG1 antisense hs.601664 and AK300104 ( FIG. 1 ).
实时PCR结果表明，在用针对MDA5反义物BU663736设计的两种寡物处理后48h，HepG2细胞中MDA5mRNA的水平显著增加(图2)。Real-time PCR results showed that the level of MDA5 mRNA in HepG2 cells was significantly increased 48 hours after treatment with two oligos designed against MDA5 antisense BU663736 ( FIG. 2 ).
实时PCR结果表明，在用针对IFNAI反义物DA393812设计的一种寡物处理后48h，HepG2细胞中IFNAI mRNA的水平显著增加(图3)。Real-time PCR results showed that the level of IFNAI mRNA in HepG2 cells was significantly increased 48 hours after treatment with an oligo designed against IFNAI antisense DA393812 ( FIG. 3 ).
用反义寡核苷酸处理ZR75细胞：Treat ZR75 cells with antisense oligonucleotides:
使来自ATCC的ZR75细胞(目录号CRL-1500)在37℃和5％CO2下生长于生长培养基(MEM/EBSS(Hyclone目录号SH30024或Mediatech目录号MT-10-010-CV)+10％FBS(Mediatech目录号MT35-011-CV)+青霉素/链霉素(Mediatech目录号MT30-002-CI))。在试验的前一天，将所述细胞以密度1.5×105/ml再接种到6孔板，并在37℃和5％CO2下培养。在试验当天，将6孔板中的培养基换成新鲜的生长培养基。将所有反义寡核苷酸稀释到20μM的浓度。将2μl此溶液与400μl Opti-MEM培养基(Gibco目录号31985-070)和4μl脂质体转染胺2000(Invitrogen目录号11668019)在室温温育20min，然后施用到具有ZR75细胞的6孔板的每个孔。含有2μl水(代替所述寡核苷酸溶液)的类似混合物用于假转染的对照。在37℃和5％CO2下培养3-18h后，将培养基换成新鲜的生长培养基。添加反义寡核苷酸48h后，将培养基移出，并遵循制造商的说明书，使用Promega的SV全RNA分离系统(目录号Z3105)或Qiagen的RNeasy全RNA分离试剂盒(目录号74181)从细胞中提取RNA。如制造商的方案中所述，向使用Thermo Scientific的Verso cDNA试剂盒(目录号AB1453B)或高容量cDNA反转录试剂盒(目录号4368813)进行的反转录反应中添加600ng RNA。将来自此反转录反应的cDNA用于通过实时PCR监测基因表达，其中使用ABI Taqman Gene Expression Mix(目录号4369510)和由ABI(Applied Biosystems Taqman Gene Expression Assay：Hs00855471_g1)设计的引物/探针。使用下面的PCR循环：50℃，2min；95℃，10min；40个(95℃，15秒；60℃，1min)循环，使用StepOnePlus Real Time PCR仪器(Applied Biosystems)。ZR75 cells from ATCC (Cat. No. CRL-1500)were grown in Growth Medium (MEM/EBSS (Hyclone Cat. No. SH30024 or Mediatech Cat. No. MT-10-010-CV) + 10 % FBS (Mediatech Cat. No. MT35-011-CV) + Penicillin/Streptomycin (Mediatech Cat. No. MT30-002-CI)). The day before the experiment, the cells were reseeded into 6-well plates at a density of 1.5×105 /ml and cultured at 37° C. and 5% CO2 . On the day of the experiment, the medium in the 6-well plate was replaced with fresh growth medium. All antisense oligonucleotides were diluted to a concentration of 20 μM. 2 μl of this solution was incubated with 400 μl of Opti-MEM medium (Gibco Cat. No. 31985-070) and 4 μl of Lipofectamine 2000 (Invitrogen Cat. No. 11668019) for 20 min at room temperature and then applied to a 6-well plate with ZR75 cells each hole. A similar mixture containing 2 μl of water (instead of the oligonucleotide solution) was used as a mock-transfected control. After 3-18 h of incubation at 37 °C and 5%CO , the medium was replaced with fresh growth medium. 48h after the addition of antisense oligonucleotides, the medium was removed, and following the manufacturer’s instructions, Promega’s SV Total RNA Isolation System (Cat. No. Z3105) or Qiagen’s RNeasy Total RNA Isolation Kit (Cat. No. 74181) was used to extract Extract RNA from cells. 600 ng of RNA was added to a reverse transcription reaction performed using Thermo Scientific's Verso cDNA Kit (Cat# AB1453B) or High Capacity cDNA Reverse Transcription Kit (Cat# 4368813) as described in the manufacturer's protocol. The cDNA from this reverse transcription reaction was used to monitor gene expression by real-time PCR using ABI Taqman Gene Expression Mix (Catalog No. 4369510) and primers/probes designed by ABI (Applied Biosystems Taqman Gene Expression Assay: Hs00855471_g1). The following PCR cycle was used: 50°C, 2 min; 95°C, 10 min; 40 cycles (95°C, 15 sec; 60°C, 1 min) using a StepOnePlus Real Time PCR instrument (Applied Biosystems).
基于处理的和假转染的样品之间18S-标准化的dCt值的不同，计算用反义寡核苷酸处理后基因表达的倍数变化。Fold changes in gene expression after treatment with antisense oligonucleotides were calculated based on the difference in 18S-normalized dCt values between treated and mock-transfected samples.
结果：实时PCR结果表明，在用针对IFNAI反义物DA393812设计的一种寡物处理后48h，ZR75细胞中IFNAI mRNA的水平显著增加(图4)。Results: The results of real-time PCR showed that the level of IFNAI mRNA in ZR75 cells was significantly increased 48 hours after treatment with an oligo designed against IFNAI antisense DA393812 ( FIG. 4 ).
用反义寡核苷酸处理HUVEC细胞Treatment of HUVEC cells with antisense oligonucleotides
使来自ATCC的HUVEC细胞(Promo Cell目录号C-12253)在37℃和5％CO2下生长于上皮生长培养基(Promo Cell目录号C-22010)。在试验的前一天，使用Promo Cell DetachKit(目录号C-41200)，将所述细胞以密度1.5×105/ml再接种到6孔板，并在37℃和5％CO2下培养。在试验当天，将6孔板中的培养基换成新鲜的上皮生长培养基。将所有反义寡核苷酸稀释到20μM的浓度。将2μl此溶液与400μlOpti-MEM培养基(Gibco目录号31985-070)和4μl脂质体转染胺2000(Invitrogen目录号11668019)在室温温育20min，然后施用到具有HUVEC细胞的6孔板的每个孔。含有2μl水(代替所述寡核苷酸溶液)的类似混合物用于假转染的对照。在37℃和5％CO2下培养3-18h后，将培养基换成新鲜的生长培养基。添加反义寡核苷酸48h后，将培养基移出，并遵循制造商的说明书，使用Promega的SV全RNA分离系统(目录号Z3105)或Qiagen的RNeasy全RNA分离试剂盒(目录号74181)从细胞中提取RNA。如制造商的方案中所述，向使用Thermo Scientific的Verso cDNA试剂盒(目录号AB1453B)进行的反转录反应中添加600ng RNA。将来自此反转录反应的cDNA用于通过实时PCR监测基因表达，其中使用ABI Taqman Gene Expression Mix(目录号4369510)和由ABI(Applied BiosystemsTaqman Gene ExpressionAssay：Hs00153340_m1和Hs00855471_g1，Applied BiosystemsInc.，Foster City CA)设计的引物/探针。使用下面的PCR循环：50℃，2min；95℃，10min；40个(95℃，15秒；60℃，1min)循环，使用StepOne Plus Real Time PCR仪器(AppliedBiosystems)或Mx4000热循环仪(Stratagene)。HUVEC cells from ATCC (Promo Cell Cat# C-12253) were grown in Epithelial Growth Medium (Promo Cell Cat# C-22010) at 37°C and 5%CO2 . The day before the experiment, the cells were reseeded into 6-well plates at a density of 1.5×105 /ml using the Promo Cell Detach Kit (Catalog No. C-41200), and cultured at 37° C. and 5% CO2 . On the day of the experiment, the medium in the 6-well plate was replaced with fresh epithelial growth medium. All antisense oligonucleotides were diluted to a concentration of 20 μM. 2 μl of this solution was incubated with 400 μl of Opti-MEM medium (Gibco catalog number 31985-070) and 4 μl of lipofectamine 2000 (Invitrogen catalog number 11668019) at room temperature for 20 min, and then applied to the 6-well plate with HUVEC cells. per hole. A similar mixture containing 2 μl of water (instead of the oligonucleotide solution) was used as a mock-transfected control. After 3-18 h of incubation at 37 °C and 5%CO , the medium was replaced with fresh growth medium. 48h after the addition of antisense oligonucleotides, the medium was removed, and following the manufacturer’s instructions, Promega’s SV Total RNA Isolation System (Cat. No. Z3105) or Qiagen’s RNeasy Total RNA Isolation Kit (Cat. No. 74181) was used to extract Extract RNA from cells. 600 ng of RNA was added to a reverse transcription reaction performed using Thermo Scientific's Verso cDNA kit (Catalog # AB1453B) as described in the manufacturer's protocol. The cDNA from this reverse transcription reaction was used to monitor gene expression by real-time PCR using the ABI Taqman Gene Expression Mix (Catalog No. 4369510) and by ABI (Applied Biosystems Taqman Gene Expression Assay: Hs00153340_m1 and Hs00855471_g1, Applied Biosystems Inc., Foster City CA ) designed primers/probes. The following PCR cycles were used: 50°C, 2min; 95°C, 10min; 40 cycles (95°C, 15 sec; 60°C, 1min) using a StepOne Plus Real Time PCR instrument (AppliedBiosystems) or a Mx4000 thermal cycler (Stratagene) .
基于处理的和假转染的样品之间18S-标准化的dCt值的不同，计算用反义寡核苷酸处理后基因表达的倍数变化。Fold changes in gene expression after treatment with antisense oligonucleotides were calculated based on the difference in 18S-normalized dCt values between treated and mock-transfected samples.
结果：实时PCR结果表明，在用针对IFNAI反义物DA393812设计的一种寡物处理后48h，HUVEC细胞中IFNAI mRNA的水平显著增加(图5)。Results: The real-time PCR results showed that the level of IFNAI mRNA in HUVEC cells was significantly increased 48 hours after treatment with an oligo designed against IFNAI antisense DA393812 ( FIG. 5 ).
用反义寡核苷酸处理MCF-7细胞：MCF-7 cells were treated with antisense oligonucleotides:
使来自ATCC的MCF-7细胞(目录号HTB-22)在37℃和5％CO2下生长于生长培养基(MEM/EBSS(Hyclone目录号SH30024或Mediatech目录号MT-10-010-CV)+10％FBS(Mediatech目录号MT35-011-CV)+青霉素/链霉素(Mediatech目录号MT30-002-CI))。在试验的前一天，将所述细胞以密度1.5×105/ml再接种到6孔板，并在37℃和5％CO2下培养。在试验当天，将6孔板中的培养基换成新鲜的生长培养基。将所有反义寡核苷酸稀释到20μM的浓度。将2μl此溶液与400μl Opti-MEM培养基(Gibco目录号31985-070)和4μl脂质体转染胺2000(Invitrogen目录号11668019)在室温温育20min，然后施用到具有MCF-7细胞的6孔板的每个孔。含有2μl水(代替所述寡核苷酸溶液)的类似混合物用于假转染的对照。在37℃和5％CO2下培养3-18h后，将培养基换成新鲜的生长培养基。添加反义寡核苷酸48h后，将培养基移出，并遵循制造商的说明书，使用Promega的SV全RNA分离系统(目录号Z3105)或Qiagen的RNeasy全RNA分离试剂盒(目录号74181)从细胞中提取RNA。如制造商的方案中所述，向使用Thermo Scientific的Verso cDNA试剂盒(目录号AB1453B)或高容量cDNA反转录试剂盒(目录号4368813)进行的反转录反应中添加600ng RNA。将来自此反转录反应的cDNA用于通过实时PCR监测基因表达，其中使用ABI Taqman Gene Expression Mix(目录号4369510)和由ABI(Applied Biosystems Taqman Gene Expression Assay：Hs00855471_g1)设计的引物/探针。使用下面的PCR循环：50℃，2min；95℃，10min；40个(95℃，15秒；60℃，1min)循环，使用StepOne Plus Real Time PCR仪器(Applied Biosystems)。MCF-7 cells from ATCC (Catalog #HTB-22) were grown in Growth Medium (MEM/EBSS (Hyclone Cat# SH30024 or Mediatech Cat# MT-10-010-CV) at 37°C and 5%CO + 10% FBS (Mediatech Cat# MT35-011-CV) + Penicillin/Streptomycin (Mediatech Cat# MT30-002-CI)). The day before the experiment, the cells were reseeded into 6-well plates at a density of 1.5×105 /ml and cultured at 37° C. and 5% CO2 . On the day of the experiment, the medium in the 6-well plate was replaced with fresh growth medium. All antisense oligonucleotides were diluted to a concentration of 20 μM. 2 μl of this solution was incubated with 400 μl of Opti-MEM medium (Gibco catalog number 31985-070) and 4 μl of lipofectamine 2000 (Invitrogen catalog number 11668019) at room temperature for 20 min, and then applied to 6 each well of the orifice plate. A similar mixture containing 2 μl of water (instead of the oligonucleotide solution) was used as a mock-transfected control. After 3-18 h of incubation at 37 °C and 5%CO , the medium was replaced with fresh growth medium. 48h after the addition of antisense oligonucleotides, the medium was removed, and following the manufacturer’s instructions, Promega’s SV Total RNA Isolation System (Cat. No. Z3105) or Qiagen’s RNeasy Total RNA Isolation Kit (Cat. No. 74181) was used to extract Extract RNA from cells. 600 ng of RNA was added to a reverse transcription reaction performed using Thermo Scientific's Verso cDNA Kit (Cat# AB1453B) or High Capacity cDNA Reverse Transcription Kit (Cat# 4368813) as described in the manufacturer's protocol. The cDNA from this reverse transcription reaction was used to monitor gene expression by real-time PCR using ABI Taqman Gene Expression Mix (Catalog No. 4369510) and primers/probes designed by ABI (Applied Biosystems Taqman Gene Expression Assay: Hs00855471_g1). The following PCR cycle was used: 50°C, 2 min; 95°C, 10 min; 40 cycles (95°C, 15 sec; 60°C, 1 min) using a StepOne Plus Real Time PCR instrument (Applied Biosystems).
基于处理的和假转染的样品之间18S-标准化的dCt值的不同，计算用反义寡核苷酸处理后基因表达的倍数变化。Fold changes in gene expression after treatment with antisense oligonucleotides were calculated based on the difference in 18S-normalized dCt values between treated and mock-transfected samples.
结果：ELISA测定结果表明，在用针对IFNAI反义物DA39381设计的寡物处理的MCF7细胞的提取物中，IFNA蛋白被增量调节(图6)。Results: ELISA assay results showed that IFNA protein was upregulated in extracts of MCF7 cells treated with oligos designed against IFNAI antisense DA39381 ( FIG. 6 ).
尽管本发明已就一个或更多个实现举例说明并描述，但在阅读和理解本说明书和附图后，本领域技术人员将想到等同改变和修饰。此外，虽然本发明的具体特征可能已就几个实现中的唯一一个公开，但这类特征可与其它实现的一个或更多个其它特征组合，因为对于任何给定或具体应用而言可为所需和有利的。While the invention has been illustrated and described with respect to one or more implementations, equivalent alterations and modifications will occur to others skilled in the art upon the reading and understanding of this specification and the annexed drawings. Furthermore, although a particular feature of the invention may be disclosed with respect to only one of several implementations, such feature may be combined with one or more other features of other implementations, as may be possible for any given or particular application. desired and beneficial.
本公开内容的摘要将允许读者快速确定本技术公开内容的性质。在理解以下的情况下将其提出：其将不用于解释或限制随附权利要求的范围或含义。The Abstract of the Disclosure will allow the reader to quickly ascertain the nature of the technical disclosure. It is submitted with the understanding that it will not be used to interpret or limit the scope or meaning of the appended claims.
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| US23145809P | 2009-08-05 | 2009-08-05 | |
| US61/231458 | 2009-08-05 | ||
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| US23788609P | 2009-08-28 | 2009-08-28 | |
| US61/237,886 | 2009-08-28 | ||
| US61/237886 | 2009-08-28 | ||
| PCT/US2010/036558WO2010138806A2 (en) | 2009-05-28 | 2010-05-28 | Treatment of antiviral gene related diseases by inhibition of natural antisense transcript to an antiviral gene |
| Publication Number | Publication Date |
|---|---|
| CN103221541A CN103221541A (en) | 2013-07-24 |
| CN103221541Btrue CN103221541B (en) | 2017-03-01 |
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201080036011.5AExpired - Fee RelatedCN103221541B (en) | 2009-05-28 | 2010-05-28 | Treatment of antiviral gene-associated diseases by inhibiting natural antisense transcripts of antiviral genes |
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| EP (1) | EP2435571B1 (en) |
| JP (1) | JP5960049B2 (en) |
| KR (2) | KR20120024819A (en) |
| CN (1) | CN103221541B (en) |
| CA (1) | CA2764683A1 (en) |
| ES (1) | ES2618576T3 (en) |
| WO (1) | WO2010138806A2 (en) |
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